Antimicrobial Resistance, Conjugative Plasmids and Pathogenicity in Escherichia spp. isolated from Natural water and Wastewater Environments in Stockholm, Sweden

Antimicrobial Resistance, Conjugative Plasmids and Pathogenicity in Escherichia spp. isolated from Natural water and Wastewater Environments in Stockholm, Sweden | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Antimicrobial Resistance, Conjugative Plasmids and Pathogenicity in Escherichia spp. isolated from Natural water and Wastewater Environments in Stockholm, Sweden Annie Justh de Neczpal, Kaisa Thorell, Laurens Tuts, Geertrui Rasschaert, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6730951/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract To investigate the occurrence of antibiotic resistance genes (ARGs), conjugative plasmids and virulence genes in Swedish waterborne Escherichia spp. isolates, water samples were collected from urban freshwater swimming areas, beaches in the Baltic Sea, and the primary wastewater treatment plant (WWTP) in Stockholm, Sweden. During the summer of 2022, a total of 69 isolates were isolated from filters grown on ESBL-selective and non-selective agar. All isolates were analyzed by phenotypic antibiotic susceptibility tests, conjugation assays and whole genome sequencing. In addition, antibiotic residues were also quantified in the water samples. Of the 69 isolates, 44 (64%) were multidrug-resistant (MDR), exhibiting resistance to three or more antibiotic classes determined by disc diffusion. WWTP-derived E. coli isolates demonstrated a significantly higher prevalence of extended-spectrum β-lactamase (ESBL) genes compared to freshwater isolates (p < 0.0001). The isolates belonged to a diverse range of multi-locus sequence types (MLST) and most isolates harbored at least one plasmid. A total of 16 strains transferred conjugative plasmids carrying various combinations of ARGs conferring resistance to cefotaxime (CTX), tetracycline, streptomycin and trimethoprim. CTX resistance genes, including bla CTX-M-15 , were found on IncF, IncN, IncBOKZ, and IncI plasmids as well as on the chromosome in a subset of ST38 isolates. IncF plasmids exhibited low (< 10 − 7 ) or undetectable transfer frequencies, while IncN and IncI1 plasmids displayed high transfer frequencies (10 − 2 − 10 − 4 ). This study demonstrates that while antimicrobial resistance in E. coli from natural water environments is still low in Stockholm, Sweden, WWTPs are a significant source of MDR and pathogenic E. coli . These strains frequently carry highly conjugative IncN and IncI1 plasmids containing multiple ARGs, posing a potential risk of increased environmental dissemination of MDR and/or pathogenic E. coli via treated effluent discharge. Biological sciences/Microbiology Earth and environmental sciences/Environmental sciences Full Text Additional Declarations No competing interests reported. Supplementary Files Table1.docx Suppl.Tables1and2.docx Suppl.figures1and2.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 18 Dec, 2025 Reviews received at journal 27 Nov, 2025 Reviewers agreed at journal 11 Nov, 2025 Reviews received at journal 25 Aug, 2025 Reviewers agreed at journal 16 Jul, 2025 Reviewers invited by journal 13 Jun, 2025 Editor assigned by journal 02 Jun, 2025 Submission checks completed at journal 26 May, 2025 First submitted to journal 23 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6730951","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":471014276,"identity":"68c940e5-dde4-43ca-aac5-a5365f72141c","order_by":0,"name":"Annie Justh de Neczpal","email":"data:image/png;base64,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","orcid":"","institution":"University of Gothenburg","correspondingAuthor":true,"prefix":"","firstName":"Annie","middleName":"Justh","lastName":"de Neczpal","suffix":""},{"id":471014277,"identity":"9a10e8b6-1002-49fc-ae88-afc1d08164b4","order_by":1,"name":"Kaisa Thorell","email":"","orcid":"","institution":"University of Gothenburg","correspondingAuthor":false,"prefix":"","firstName":"Kaisa","middleName":"","lastName":"Thorell","suffix":""},{"id":471014278,"identity":"0b66c976-ebf0-46c9-bc65-ec1c47d179cf","order_by":2,"name":"Laurens Tuts","email":"","orcid":"","institution":"Flanders Research Institute for Agriculture, Fisheries and Food (ILVO)","correspondingAuthor":false,"prefix":"","firstName":"Laurens","middleName":"","lastName":"Tuts","suffix":""},{"id":471014279,"identity":"84a2673f-a3a8-43a2-8b08-2ced50006e5f","order_by":3,"name":"Geertrui Rasschaert","email":"","orcid":"","institution":"Flanders Research Institute for Agriculture, Fisheries and Food (ILVO)","correspondingAuthor":false,"prefix":"","firstName":"Geertrui","middleName":"","lastName":"Rasschaert","suffix":""},{"id":471014281,"identity":"24a0d089-5716-4e54-ac98-b3d47d95129e","order_by":4,"name":"Alberto J. 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During the summer of 2022, a total of 69 isolates were isolated from filters grown on ESBL-selective and non-selective agar. All isolates were analyzed by phenotypic antibiotic susceptibility tests, conjugation assays and whole genome sequencing. In addition, antibiotic residues were also quantified in the water samples.\u003c/p\u003e \u003cp\u003eOf the 69 isolates, 44 (64%) were multidrug-resistant (MDR), exhibiting resistance to three or more antibiotic classes determined by disc diffusion. WWTP-derived \u003cem\u003eE. coli\u003c/em\u003e isolates demonstrated a significantly higher prevalence of extended-spectrum β-lactamase (ESBL) genes compared to freshwater isolates (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The isolates belonged to a diverse range of multi-locus sequence types (MLST) and most isolates harbored at least one plasmid. A total of 16 strains transferred conjugative plasmids carrying various combinations of ARGs conferring resistance to cefotaxime (CTX), tetracycline, streptomycin and trimethoprim. CTX resistance genes, including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-15\u003c/sub\u003e, were found on IncF, IncN, IncBOKZ, and IncI plasmids as well as on the chromosome in a subset of ST38 isolates. IncF plasmids exhibited low (\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e) or undetectable transfer frequencies, while IncN and IncI1 plasmids displayed high transfer frequencies (10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e \u0026minus;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e). This study demonstrates that while antimicrobial resistance in \u003cem\u003eE. coli\u003c/em\u003e from natural water environments is still low in Stockholm, Sweden, WWTPs are a significant source of MDR and pathogenic \u003cem\u003eE. coli\u003c/em\u003e. These strains frequently carry highly conjugative IncN and IncI1 plasmids containing multiple ARGs, posing a potential risk of increased environmental dissemination of MDR and/or pathogenic \u003cem\u003eE. coli\u003c/em\u003e via treated effluent discharge.\u003c/p\u003e","manuscriptTitle":"Antimicrobial Resistance, Conjugative Plasmids and Pathogenicity in Escherichia spp. isolated from Natural water and Wastewater Environments in Stockholm, Sweden","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 10:14:40","doi":"10.21203/rs.3.rs-6730951/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-18T16:25:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-28T03:59:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159712374824637712064163368761655644116","date":"2025-11-12T00:17:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-25T07:40:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151672108625579738992711067302900367418","date":"2025-07-16T11:29:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-13T16:04:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-02T18:10:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-26T07:17:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Antimicrobials and Resistance","date":"2025-05-23T08:24:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-antimicrobials-and-resistance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjamar","sideBox":"Learn more about [npj Antimicrobials and Resistance](http://www.nature.com/npjamar/)","snPcode":"44259","submissionUrl":"https://submission.springernature.com/new-submission/44259/3","title":"npj Antimicrobials and Resistance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3c1b716a-18a7-44ce-b3e1-a6264b6325be","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":50023824,"name":"Biological sciences/Microbiology"},{"id":50023825,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2026-04-14T18:09:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-17 10:14:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6730951","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6730951","identity":"rs-6730951","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}