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AIEC are genetically diverse, and in the absence of a universal molecular signature, are defined by\n                    <jats:italic>in vitro<\/jats:italic>\n                    functional attributes. The relative ability of difference AIEC strains to colonize, persist, and induce inflammation in an IBD-susceptible host is unresolved. To evaluate strain-level variation among tissue-associated\n                    <jats:italic>E<\/jats:italic>\n                    .\n                    <jats:italic>coli<\/jats:italic>\n                    in the intestines, we develop a long-read sequencing approach to identify AIEC by strain that excludes host DNA. We use this approach to distinguish genetically similar strains and assess their fitness in colonizing the intestine. Here we have assembled complete genomes using long-read nanopore sequencing for a model AIEC strain, NC101, and seven strains isolated from the intestinal mucosa of Crohn\u2019s disease and non-Crohn\u2019s tissues. We show these strains can colonize the intestine of IBD susceptible mice and induce inflammatory cytokines from cultured macrophages. We demonstrate that these strains can be quantified and distinguished in the presence of 99.5% mammalian DNA and from within a fecal population. Analysis of global genomic structure and specific sequence variation within the ribosomal RNA operon provides a framework for efficiently tracking strain-level variation of closely-related\n                    <jats:italic>E<\/jats:italic>\n                    .\n                    <jats:italic>coli<\/jats:italic>\n                    and likely other commensal\/pathogenic bacteria impacting intestinal inflammation in experimental settings and IBD patients.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0259141","type":"journal-article","created":{"date-parts":[[2021,10,28]],"date-time":"2021-10-28T13:48:31Z","timestamp":1635428911000},"page":"e0259141","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":12,"title":["Long-read sequencing to interrogate strain-level variation among adherent-invasive Escherichia coli isolated from human intestinal tissue"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0673-9418","authenticated-orcid":true,"given":"Jeremy","family":"Wang","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rachel M.","family":"Bleich","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sandra","family":"Zarmer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shiying","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Belgin","family":"Dogan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kenneth W.","family":"Simpson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4796-0207","authenticated-orcid":true,"given":"Janelle C.","family":"Arthur","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2021,10,28]]},"reference":[{"issue":"2","key":"pone.0259141.ref001","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1053\/j.gastro.2016.10.012","article-title":"Roles for Intestinal Bacteria, Viruses, and Fungi in Pathogenesis of Inflammatory Bowel Diseases and Therapeutic Approaches","volume":"152","author":"RB Sartor","year":"2017","journal-title":"Gastroenterology"},{"issue":"2","key":"pone.0259141.ref002","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/j.cgh.2013.03.034","article-title":"Risk of cancer in patients with inflammatory bowel diseases: A nationwide population-based cohort study with 30 years of follow-up evaluation","volume":"12","author":"MD Kappelman","year":"2014","journal-title":"Clin Gastroenterol Hepatol"},{"issue":"6103","key":"pone.0259141.ref003","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1126\/science.1224820","article-title":"Intestinal Inflammation Targets Cancer-Inducing Activity of the Microbiota","volume":"338","author":"JC Arthur","year":"2012","journal-title":"Science"},{"issue":"6375","key":"pone.0259141.ref004","doi-asserted-by":"crossref","first-page":"592","DOI":"10.1126\/science.aah3648","article-title":"Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria","volume":"359","author":"CM Dejea","year":"2018","journal-title":"Science"},{"issue":"2","key":"pone.0259141.ref005","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1053\/j.gastro.2004.04.061","article-title":"High Prevalence of Adherent-Invasive Escherichia Coli Associated With Ileal Mucosa in Crohn\u2019s Disease","volume":"127","author":"A Darfeuille-Michaud","year":"2004","journal-title":"Gastroenterology"},{"issue":"1","key":"pone.0259141.ref006","first-page":"80","article-title":"Enhanced Escherichia coli adherence and invasion in Crohn\u2019s disease and colon cancer","volume":"127","author":"HM Martin","year":"2004","journal-title":"Gastroenterology"},{"issue":"5","key":"pone.0259141.ref007","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1038\/ismej.2007.52","article-title":"Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn\u2019s disease involving the ileum.","volume":"1","author":"M Baumgart","year":"2007","journal-title":"ISME J"},{"issue":"10","key":"pone.0259141.ref008","doi-asserted-by":"crossref","first-page":"1505","DOI":"10.1136\/gutjnl-2012-303954","article-title":"Advances in inflammatory bowel disease pathogenesis: Linking host genetics and the microbiome","volume":"62","author":"D Knights","year":"2013","journal-title":"Gut"},{"issue":"34","key":"pone.0259141.ref009","doi-asserted-by":"crossref","first-page":"13780","DOI":"10.1073\/pnas.0706625104","article-title":"Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases","volume":"104","author":"DN Frank","year":"2007","journal-title":"Proc Natl Acad Sci U S A"},{"key":"pone.0259141.ref010","doi-asserted-by":"crossref","first-page":"1403","DOI":"10.1038\/ismej.2014.3","article-title":"Gut microbiome composition and function in experimental colitis during active disease and treatment-induced remission","volume":"8","author":"MG Rooks","year":"2014","journal-title":"ISME J"},{"issue":"472","key":"pone.0259141.ref011","doi-asserted-by":"crossref","first-page":"eaap8914","DOI":"10.1126\/scitranslmed.aap8914","article-title":"Gut microbiota composition and functional changes in inflammatory bowel disease and irritable bowel 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in TLR5-deficient mice.","volume":"12","author":"FA Carvalho","year":"2012","journal-title":"Cell Host Microbe."},{"issue":"8","key":"pone.0259141.ref015","doi-asserted-by":"crossref","first-page":"1382","DOI":"10.1136\/gutjnl-2015-311059","article-title":"Comparative genomics of Crohn\u2019s disease-Associated adherent-invasive Escherichia coli","volume":"66","author":"CL O\u2019Brien","year":"2017","journal-title":"Gut"},{"issue":"1","key":"pone.0259141.ref016","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1002\/ibd.22971","article-title":"Multidrug Resistance Is Common in Escherichia coli Associated with Ileal Crohn\u2019s Disease.","volume":"19","author":"B Dogan","year":"2013","journal-title":"Inflamm Bowel Dis."},{"issue":"1","key":"pone.0259141.ref017","doi-asserted-by":"crossref","DOI":"10.1186\/s13742-016-0111-z","article-title":"Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION\u2122 portable nanopore 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Tyson","year":"2020"},{"issue":"14","key":"pone.0259141.ref024","doi-asserted-by":"crossref","first-page":"2103","DOI":"10.1093\/bioinformatics\/btw152","article-title":"Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences.","volume":"32","author":"H Li","year":"2016","journal-title":"Bioinformatics"},{"key":"pone.0259141.ref025","doi-asserted-by":"crossref","first-page":"737","DOI":"10.1101\/gr.214270.116","article-title":"Fast and accurate de novo genome assembly from long uncorrected reads","volume":"27","author":"R Vaser","year":"2017","journal-title":"Genome Res"},{"key":"pone.0259141.ref026","unstructured":"Oxford Nanopore Technologies. Medaka; 2018 [cited 2020 March 3]. Available from: https:\/\/nanoporetech.github.io\/medaka\/."},{"issue":"11","key":"pone.0259141.ref027","doi-asserted-by":"crossref","first-page":"e112963","DOI":"10.1371\/journal.pone.0112963","article-title":"Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement.","volume":"9","author":"BJ Walker","year":"2014","journal-title":"PLOS ONE"},{"issue":"14","key":"pone.0259141.ref028","doi-asserted-by":"crossref","first-page":"6614","DOI":"10.1093\/nar\/gkw569","article-title":"NCBI prokaryotic genome annotation pipeline","volume":"44","author":"T Tatusova","year":"2016","journal-title":"Nucleic Acids Res"},{"key":"pone.0259141.ref029","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1038\/nmeth.3176","article-title":"Fast and sensitive protein alignment using DIAMOND","volume":"12","author":"B Buchfink","year":"2015","journal-title":"Nature Methods"},{"issue":"11","key":"pone.0259141.ref030","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1186\/s13073-014-0090-6","article-title":"SRST2: Rapid genomic surveillance for public health and hospital microbiology labs","volume":"6","author":"M Inouye","year":"2014","journal-title":"Genome Med"},{"key":"pone.0259141.ref031","unstructured":"Center for Genomic Epidemiology; 2011 [cited 2020 March 3]. 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Li","year":"2018","journal-title":"Bioinformatics"},{"issue":"1","key":"pone.0259141.ref033","doi-asserted-by":"crossref","first-page":"mgen000490","DOI":"10.1099\/mgen.0.000490","article-title":"Genomic epidemiology of Escherichia coli isolates from a tertiary referral center in Lilongwe, Malawi.","volume":"7","author":"G Tegha","year":"2021","journal-title":"Microbial Genomics"},{"issue":"1","key":"pone.0259141.ref034","doi-asserted-by":"crossref","first-page":"e00303","DOI":"10.1128\/mBio.00303-11","article-title":"Kinetics of Uropathogenic Escherichia Coli Metapopulation Movement During Urinary Tract Infection.","volume":"3","author":"MS Walters","year":"2012","journal-title":"mBio"},{"issue":"1","key":"pone.0259141.ref035","doi-asserted-by":"crossref","first-page":"e1004587","DOI":"10.1371\/journal.ppat.1004587","article-title":"Dissemination of a highly virulent pathogen: tracking the early events that define infection.","volume":"11","author":"RJ Gonzalez","year":"2015","journal-title":"PLoS Pathog."},{"issue":"4724","key":"pone.0259141.ref036","article-title":"Microbial genomic analysis reveals the essential role of inflammation in bacteria-induced colorectal cancer","volume":"5","author":"JC Arthur","year":"2014","journal-title":"Nature Communications"},{"issue":"2","key":"pone.0259141.ref037","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1002\/j.1538-7305.1950.tb00463.x","article-title":"Error detecting and error correcting codes","volume":"29","author":"RW Hamming","year":"1950","journal-title":"The Bell System Technical Journal"},{"issue":"2","key":"pone.0259141.ref038","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1016\/0092-8674(93)80068-P","article-title":"Interleukin-10-deficient mice develop chronic enterocolitis","volume":"75","author":"R 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Patwa","year":"2011","journal-title":"Gastroenterology"},{"issue":"10","key":"pone.0259141.ref042","doi-asserted-by":"crossref","first-page":"4068","DOI":"10.1128\/IAI.00904-15","article-title":"Adherent-invasive Escherichia coli production of cellulose influences iron-induced bacterial aggregation, phagocytosis, and induction of colitis","volume":"83","author":"M Ellermann","year":"2015","journal-title":"Infect Immun"},{"issue":"3","key":"pone.0259141.ref043","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1136\/gut.47.3.397","article-title":"Butyrate inhibits inflammatory responses through NF\u03baB inhibition: Implications for Crohn\u2019s disease","volume":"47","author":"JP Segain","year":"2000","journal-title":"Gut"},{"issue":"2","key":"pone.0259141.ref044","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1016\/j.chom.2018.12.010","article-title":"Host-Specific Adaptive Diversification of Crohn\u2019s Escherichia coli Article Host-Specific Adaptive Diversification of Crohn\u2019s Disease-Associated Adherent-Invasive Escherichia coli.","volume":"25","author":"W Elhenawy","year":"2019","journal-title":"Cell Host Microbe"},{"issue":"20212032","key":"pone.0259141.ref045","article-title":"High-throughput fitness screening and transcriptomics identify a role for a type IV secretion system in the pathogenesis of Crohn\u00e2\u20acTMs disease-associated Escherichia coli.","volume":"12","author":"W Elhenawy","journal-title":"Nat Commun."},{"issue":"5","key":"pone.0259141.ref046","doi-asserted-by":"crossref","first-page":"1272","DOI":"10.1111\/j.1365-2958.2001.02315.x","article-title":"Type 1 pili-mediated adherence of Escherichia coli strain LF82 isolated from Crohn\u2019s disease is involved in bacterial invasion of intestinal epithelial cells.","volume":"39","author":"\u00c3 Boudeau","year":"2001","journal-title":"Mol 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