{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T01:15:49Z","timestamp":1783646149546,"version":"3.55.0"},"reference-count":47,"publisher":"Oxford University Press (OUP)","license":[{"start":{"date-parts":[[2019,10,21]],"date-time":"2019-10-21T00:00:00Z","timestamp":1571616000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004359","name":"Swedish Research Council","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004359","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Erasmus Mundus Joint Master Degree"},{"name":"EMJMD"},{"name":"Erasmus+ EU-Programme"},{"name":"International Master in Innovative Medicine (IMIM) programme"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Objectives<\/jats:title><jats:p>To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX)\u202f=\u202f16\u2009mg\/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX\u202f=\u202f32\u201364\u2009mg\/L) had mutations in the emrR gene, together with either a 50\u2009kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.<\/jats:p><\/jats:sec>","DOI":"10.1093\/jac\/dkz434","type":"journal-article","created":{"date-parts":[[2019,10,1]],"date-time":"2019-10-01T19:27:12Z","timestamp":1569958032000},"source":"Crossref","is-referenced-by-count":24,"title":["Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coli"],"prefix":"10.1093","author":[{"given":"Fabiola","family":"Pu\u00e9rtolas-Balint","sequence":"first","affiliation":[{"name":"Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123, Uppsala, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Omar","family":"Warsi","sequence":"additional","affiliation":[{"name":"Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123, Uppsala, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marius","family":"Linkevicius","sequence":"additional","affiliation":[{"name":"Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123, Uppsala, Sweden"},{"name":"Department of Public Health Solutions, National Institute for Health and Welfare (THL), Helsinki, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5642-0360","authenticated-orcid":false,"given":"Po-Cheng","family":"Tang","sequence":"additional","affiliation":[{"name":"Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123, Uppsala, Sweden"},{"name":"Department of Medical Cell Biology, Uppsala University, SE-75123, Uppsala, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dan I","family":"Andersson","sequence":"additional","affiliation":[{"name":"Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123, Uppsala, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2019,10,21]]},"reference":[{"key":"2019102109591516800_dkz434-B1","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1097\/MOU.0000000000000365","article-title":"The revival of old antibiotics for treatment of uncomplicated urinary tract infections in the era of antibiotic stewardship","volume":"27","author":"Kranz","year":"2017","journal-title":"Curr Opin Urol"},{"key":"2019102109591516800_dkz434-B2","doi-asserted-by":"crossref","first-page":"2916","DOI":"10.1093\/jac\/dky255","article-title":"Review of the pharmacokinetic properties of nitrofurantoin and nitroxoline","volume":"73","author":"Wijma","year":"2018","journal-title":"J Antimicrob Chemother"},{"key":"2019102109591516800_dkz434-B3","first-page":"406","article-title":"Microbiological consequences of chelation of bivalent metal cations by nitroxoline]","volume":"42","author":"Pelletier","year":"1994","journal-title":"Pathol Biol (Paris)"},{"key":"2019102109591516800_dkz434-B4","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1128\/AAC.39.3.707","article-title":"Roles of divalent cations and pH in mechanism of action of nitroxoline against Escherichia coli strains","volume":"39","author":"Pelletier","year":"1995","journal-title":"Antimicrob Agents Chemother"},{"key":"2019102109591516800_dkz434-B5","doi-asserted-by":"crossref","first-page":"6021","DOI":"10.1128\/AAC.01484-12","article-title":"The urinary antibiotic 5-nitro-8-hydroxyquinoline (nitroxoline) reduces the formation and induces the dispersal of Pseudomonas aeruginosa biofilms by chelation of iron and zinc","volume":"56","author":"Sobke","year":"2012","journal-title":"Antimicrob Agents Chemother"},{"key":"2019102109591516800_dkz434-B6","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1042\/bj1470401","article-title":"The mechanism of inhibition of ribonucleic acid synthesis by 8-hydroxyquinoline and the antibiotic lomofungin","volume":"147","author":"Fraser","year":"1975","journal-title":"Biochem J"},{"key":"2019102109591516800_dkz434-B7","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1016\/j.ijantimicag.2016.10.017","article-title":"Nitroxoline: a broad-spectrum biofilm-eradicating agent against pathogenic bacteria","volume":"49","author":"Abouelhassan","year":"2017","journal-title":"Int J Antimicrob Agents"},{"key":"2019102109591516800_dkz434-B8","first-page":"455","article-title":"[Direct effect of nitroxoline in the inhibition of bacterial adherence to urinary catheters]","volume":"38","author":"Oliviero","year":"1990","journal-title":"Pathol Biol (Paris)"},{"key":"2019102109591516800_dkz434-B9","doi-asserted-by":"crossref","first-page":"7019","DOI":"10.1128\/AAC.03946-14","article-title":"In vitro activity of nitroxoline against Escherichia coli urine isolates from outpatient departments in Germany","volume":"58","author":"Kresken","year":"2014","journal-title":"Antimicrob Agents Chemother"},{"key":"2019102109591516800_dkz434-B10","first-page":"452","article-title":"Inhibition of bacterial adherence by nitroxoline on cellular adhesion and on urinary catheter surfaces]","volume":"36","author":"Karam","year":"1988","journal-title":"Pathol Biol"},{"key":"2019102109591516800_dkz434-B11","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1159\/000487645","article-title":"The 2017 update of the German clinical guideline on epidemiology, diagnostics, therapy, prevention, and management of uncomplicated urinary tract infections in adult patients. Part II: therapy and prevention","volume":"100","author":"Kranz","year":"2018","journal-title":"Urol Int"},{"key":"2019102109591516800_dkz434-B12","doi-asserted-by":"crossref","first-page":"628.","DOI":"10.1186\/s12879-014-0628-7","article-title":"Review of the literature and individual patients\u2019 data meta-analysis on efficacy and tolerance of nitroxoline in the treatment of uncomplicated urinary tract infections","volume":"14","author":"Naber","year":"2014","journal-title":"BMC Infect Dis"},{"key":"2019102109591516800_dkz434-B13","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.ijantimicag.2017.10.010","article-title":"Empirical treatment of lower urinary tract infections in the face of spreading multidrug resistance: in vitro study on the effectiveness of nitroxoline","volume":"51","author":"Sobke","year":"2018","journal-title":"Int J Antimicrob Agents"},{"key":"2019102109591516800_dkz434-B14","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/j.bbrep.2016.03.014","article-title":"Derivatives (halogen, nitro and amino) of 8-hydroxyquinoline with highly potent antimicrobial and antioxidant activities","volume":"6","author":"Cherdtrakulkiat","year":"2016","journal-title":"Biochem Biophys Reports"},{"key":"2019102109591516800_dkz434-B15","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1111\/j.1439-0507.1991.tb00669.x","article-title":"In vitro activity of nitroxoline against clinical isolates of Candida species","volume":"34","author":"Hern\u00e1ndez Molina","year":"1991","journal-title":"Mycoses"},{"key":"2019102109591516800_dkz434-B16","first-page":"669","article-title":"[Clinical study and effect of nitroxoline on fecal flora in children]","volume":"35","author":"Lambert-Zechovsky","year":"1987","journal-title":"Pathol Biol (Paris)"},{"key":"2019102109591516800_dkz434-B17","doi-asserted-by":"crossref","first-page":"434","DOI":"10.1016\/j.cmi.2017.11.007","article-title":"Nitroxoline in geriatric patients with lower urinary tract infection fails to achieve microbiologic eradication: a noncomparative, prospective observational study","volume":"24","author":"Forstner","year":"2018","journal-title":"Clin Microbiol Infect"},{"key":"2019102109591516800_dkz434-B18","doi-asserted-by":"crossref","first-page":"713","DOI":"10.1128\/AAC.02147-13","article-title":"Urinary concentrations and antibacterial activities of nitroxoline at 250 milligrams versus trimethoprim at 200 milligrams against uropathogens in healthy volunteers","volume":"58","author":"Wagenlehner","year":"2014","journal-title":"Antimicrob Agents Chemother"},{"key":"2019102109591516800_dkz434-B19","doi-asserted-by":"crossref","first-page":"e33.","DOI":"10.1093\/nar\/gkw1078","article-title":"Duplication-insertion recombineering: a fast and scar-free method for efficient transfer of multiple mutations in bacteria","volume":"45","author":"N\u00e4svall","year":"2017","journal-title":"Nucleic Acids Res"},{"key":"2019102109591516800_dkz434-B20","first-page":"1","year":"2019"},{"key":"2019102109591516800_dkz434-B21","doi-asserted-by":"crossref","first-page":"865","DOI":"10.1016\/j.cmi.2017.11.020","article-title":"Antimicrobial susceptibility testing of colistin \u2013 evaluation of seven commercial MIC products against standard broth microdilution for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter spp","volume":"24","author":"Matuschek","year":"2018","journal-title":"Clin Microbiol Infect"},{"key":"2019102109591516800_dkz434-B22","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1093\/dnares\/dsi012","article-title":"Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research","volume":"12","author":"Kitagawa","year":"2005","journal-title":"DNA Res"},{"key":"2019102109591516800_dkz434-B23","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1007\/978-1-4939-0554-6_12","article-title":"Identification of mutations in laboratory evolved microbes from next-generation sequencing data using breseq","volume":"1151","author":"Deatherage","year":"2014","journal-title":"Methods Mol Biol"},{"key":"2019102109591516800_dkz434-B24","doi-asserted-by":"crossref","first-page":"8938","DOI":"10.1073\/pnas.89.19.8938","article-title":"Emr, an Escherichia coli locus for multidrug resistance","volume":"89","author":"Lomovskaya","year":"1992","journal-title":"Proc Natl Acad Sci USA"},{"key":"2019102109591516800_dkz434-B25","doi-asserted-by":"crossref","first-page":"2328","DOI":"10.1128\/jb.177.9.2328-2334.1995","article-title":"EmrR is a negative regulator of the Escherichia coli multidrug resistance pump EmrAB","volume":"177","author":"Lomovskaya","year":"1995","journal-title":"J Bacteriol"},{"key":"2019102109591516800_dkz434-B26","doi-asserted-by":"crossref","first-page":"2905","DOI":"10.1128\/AAC.44.10.2905-2907.2000","article-title":"The EmrR protein represses the Escherichia coli emrRAB multidrug resistance operon by directly binding to its promoter region","volume":"44","author":"Xiong","year":"2000","journal-title":"Antimicrob Agents Chemother"},{"key":"2019102109591516800_dkz434-B27","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3389\/fmicb.2018.02756","article-title":"EmrR-dependent upregulation of the efflux pump EmrCAB contributes to antibiotic resistance in Chromobacterium violaceum","volume":"9","author":"Barroso","year":"2018","journal-title":"Front Microbiol"},{"key":"2019102109591516800_dkz434-B28","doi-asserted-by":"crossref","first-page":"3723","DOI":"10.1128\/jb.175.12.3723-3729.1993","article-title":"Thiolactomycin resistance in Escherichia coli is associated with the multidrug resistance efflux pump encoded by emrAB","volume":"175","author":"Furukawa","year":"1993","journal-title":"J Bacteriol"},{"key":"2019102109591516800_dkz434-B29","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1128\/AAC.01048-06","article-title":"Substrate competition studies using whole-cell accumulation assays with the major tripartite multidrug efflux pumps of Escherichia coli","volume":"51","author":"Elkins","year":"2007","journal-title":"Antimicrob Agents Chemother"},{"key":"2019102109591516800_dkz434-B30","doi-asserted-by":"crossref","first-page":"2125","DOI":"10.1128\/AAC.01420-09","article-title":"Many chromosomal genes modulate MarA-mediated multidrug resistance in Escherichia coli","volume":"54","author":"Ruiz","year":"2010","journal-title":"Antimicrob Agents Chemother"},{"key":"2019102109591516800_dkz434-B31","doi-asserted-by":"crossref","first-page":"1233","DOI":"10.1111\/mmi.12429","article-title":"Lon protease inactivation, or translocation of the lon gene, potentiate bacterial evolution to antibiotic resistance","volume":"90","author":"Nicoloff","year":"2013","journal-title":"Mol Microbiol"},{"key":"2019102109591516800_dkz434-B32","doi-asserted-by":"crossref","first-page":"3467","DOI":"10.1128\/JB.182.12.3467-3474.2000","article-title":"Differential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA","volume":"182","author":"Barbosa","year":"2000","journal-title":"J Bacteriol"},{"key":"2019102109591516800_dkz434-B33","doi-asserted-by":"crossref","first-page":"3890","DOI":"10.1128\/JB.183.13.3890-3902.2001","article-title":"Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate","volume":"183","author":"Pomposiello","year":"2001","journal-title":"J Bacteriol"},{"key":"2019102109591516800_dkz434-B34","doi-asserted-by":"crossref","first-page":"2067","DOI":"10.1128\/AAC.41.10.2067","article-title":"Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon","volume":"41","author":"Alekshun","year":"1997","journal-title":"Antimicrob Agents Chemother"},{"key":"2019102109591516800_dkz434-B35","doi-asserted-by":"crossref","first-page":"1801","DOI":"10.1046\/j.1365-2958.2003.03952.x","article-title":"Proteolytic degradation of Escherichia coli transcription activators SoxS and MarA as the mechanism for reversing the induction of the superoxide (SoxRS) and multiple antibiotic resistance (Mar) regulons","volume":"51","author":"Griffith","year":"2004","journal-title":"Mol Microbiol"},{"key":"2019102109591516800_dkz434-B36","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1093\/jac\/dkn222","article-title":"Nitrofurantoin resistance mechanism and fitness cost in Escherichia coli","volume":"62","author":"Sandegren","year":"2008","journal-title":"J Antimicrob Chemother"},{"key":"2019102109591516800_dkz434-B37","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1016\/j.resmic.2018.05.003","article-title":"Structure and mechanism of bacterial tripartite efflux pumps","volume":"169","author":"Neuberger","year":"2018","journal-title":"Res Microbiol"},{"key":"2019102109591516800_dkz434-B38","doi-asserted-by":"crossref","first-page":"338","DOI":"10.1016\/j.bbrc.2009.01.081","article-title":"The multidrug resistance efflux complex, EmrAB from Escherichia coli forms a dimer in vitro","volume":"380","author":"Tanabe","year":"2009","journal-title":"Biochem Biophys Res Commun"},{"key":"2019102109591516800_dkz434-B39","doi-asserted-by":"crossref","first-page":"5131","DOI":"10.1128\/JB.181.16.5131-5133.1999","article-title":"Purification and ligand binding of EmrR, a regulator of a multidrug transporter","volume":"181","author":"Brooun","year":"1999","journal-title":"J Bacteriol"},{"key":"2019102109591516800_dkz434-B40","doi-asserted-by":"crossref","first-page":"5803","DOI":"10.1128\/JB.183.20.5803-5812.2001","article-title":"Analysis of a complete library of putative drug transporter genes in Escherichia coli","volume":"183","author":"Nishino","year":"2001","journal-title":"J Bacteriol"},{"key":"2019102109591516800_dkz434-B41","doi-asserted-by":"crossref","first-page":"2809","DOI":"10.1093\/jac\/dkt263","article-title":"Mechanisms and fitness costs of tigecycline resistance in Escherichia coli","volume":"68","author":"Linkevicius","year":"2013","journal-title":"J Antimicrob Chemother"},{"key":"2019102109591516800_dkz434-B42","doi-asserted-by":"crossref","first-page":"3699","DOI":"10.1128\/JB.184.13.3699-3703.2002","article-title":"Toxic waste disposal in Escherichia coli","volume":"184","author":"Helling","year":"2002","journal-title":"J Bacteriol"},{"key":"2019102109591516800_dkz434-B43","doi-asserted-by":"crossref","first-page":"1126","DOI":"10.1128\/AAC.45.4.1126-1136.2001","article-title":"Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes","volume":"45","author":"Sulavik","year":"2001","journal-title":"Antimicrob Agents Chemother"},{"key":"2019102109591516800_dkz434-B44","doi-asserted-by":"crossref","first-page":"2498","DOI":"10.1093\/jac\/dkt250","article-title":"Characterization of a major facilitator superfamily (MFS) tripartite efflux pump EmrCABsm from Stenotrophomonas maltophilia","volume":"68","author":"Huang","year":"2013","journal-title":"J Antimicrob Chemother"},{"key":"2019102109591516800_dkz434-B45","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1046\/j.1365-2958.1998.00657.x","article-title":"Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae","volume":"27","author":"Colmer","year":"1998","journal-title":"Mol Microbiol"},{"key":"2019102109591516800_dkz434-B46","doi-asserted-by":"crossref","first-page":"2850","DOI":"10.1128\/AAC.47.9.2850-2858.2003","article-title":"Biological costs and mechanisms of fosfomycin resistance in Escherichia coli","volume":"47","author":"Nilsson","year":"2003","journal-title":"Antimicrob Agents Chemother"},{"key":"2019102109591516800_dkz434-B47","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1111\/j.1365-2958.1992.tb01500.x","article-title":"A theoretical and experimental analysis of bacterial growth in the bladder","volume":"6","author":"Gordon","year":"1992","journal-title":"Mol Microbiol"}],"container-title":["Journal of Antimicrobial Chemotherapy"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/academic.oup.com\/jac\/advance-article-pdf\/doi\/10.1093\/jac\/dkz434\/30251989\/dkz434.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"http:\/\/academic.oup.com\/jac\/advance-article-pdf\/doi\/10.1093\/jac\/dkz434\/30251989\/dkz434.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,1,23]],"date-time":"2021-01-23T23:33:49Z","timestamp":1611444829000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/jac\/advance-article\/doi\/10.1093\/jac\/dkz434\/5601609"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,10,21]]},"references-count":47,"URL":"https:\/\/doi.org\/10.1093\/jac\/dkz434","relation":{},"ISSN":["0305-7453","1460-2091"],"issn-type":[{"value":"0305-7453","type":"print"},{"value":"1460-2091","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,10,21]]},"article-number":"dkz434"}}