{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T21:39:55Z","timestamp":1783978795559,"version":"3.55.0"},"reference-count":46,"publisher":"American Society for Microbiology","issue":"1","license":[{"start":{"date-parts":[[2025,1,7]],"date-time":"2025-01-07T00:00:00Z","timestamp":1736208000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,1,7]],"date-time":"2025-01-07T00:00:00Z","timestamp":1736208000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100014013","name":"UK Research and Innovation","doi-asserted-by":"crossref","award":["107136"],"award-info":[{"award-number":["107136"]}],"id":[{"id":"10.13039\/100014013","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100007190","name":"Unilever","doi-asserted-by":"crossref","award":["MA-2021-00523N"],"award-info":[{"award-number":["MA-2021-00523N"]}],"id":[{"id":"10.13039\/100007190","id-type":"DOI","asserted-by":"crossref"}]},{"name":"NIHR Global Health Professorship","award":["NIHR301627"],"award-info":[{"award-number":["NIHR301627"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2025,1,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Understanding transmission pathways of important opportunistic, drug-resistant pathogens, such as extended-spectrum beta-lactamase (ESBL)-producing\n                      <jats:italic>Escherichia coli<\/jats:italic>\n                      , is essential to implementing targeted prevention strategies to interrupt transmission and reduce the number of infections. To link transmission of ESBL-producing\n                      <jats:italic>E. coli<\/jats:italic>\n                      (ESBL-EC) between two sources, single-nucleotide resolution of\n                      <jats:italic>E. coli<\/jats:italic>\n                      strains, as well as\n                      <jats:italic>E. coli<\/jats:italic>\n                      diversity within and between samples, is required. However, the microbiological methods to best track these pathogens are unclear. Here, we compared different steps in the microbiological workflow to determine the impact different pre-enrichment broths, pre-enrichment incubation times, selection in pre-enrichment, selective plating, and DNA extraction methods had on recovering ESBL-EC from human stool samples, with the aim to acquire high-quality DNA for sequencing and genomic epidemiology. We demonstrate that using a 4-h pre-enrichment in Buffered Peptone Water, plating on cefotaxime-supplemented MacConkey agar and extracting DNA using Lucigen MasterPure DNA Purification kit improves the recovery of ESBL-EC from human stool and produced high-quality DNA for whole-genome sequencing. We conclude that our optimized workflow can be applied for single-nucleotide variant analysis of an ESBL-EC from stool.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Drug-resistant infections are increasingly difficult to treat with antibiotics. Preventing infections is thus highly beneficial. To do this, we need to understand how drug-resistant bacteria spread to take action to stop infection and transmission. This requires us to accurately trace these bacteria between different sources. In this study, we compared different laboratory methods to see which worked best for detecting extended-spectrum beta-lactamase (ESBL)-producing\n                        <jats:italic>E. coli<\/jats:italic>\n                        , a common cause of urinary tract or bloodstream infections, from human stool samples. We found that enriching stool in a nutrient broth for 4 h, then plating the bacterial suspension on antibiotic-selective MacConkey agar, and finally extracting DNA from the bacteria using a specific DNA purification kit resulted in improved recovery of ESBL\n                        <jats:italic>E. coli<\/jats:italic>\n                        and high-quality DNA. Sequencing multiple isolates from stool allowed us to distinguish unambiguously and at high resolution between different variants of ESBL\n                        <jats:italic>E. coli<\/jats:italic>\n                        present in stool.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.01058-24","type":"journal-article","created":{"date-parts":[[2024,11,22]],"date-time":"2024-11-22T09:01:04Z","timestamp":1732266064000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":10,"title":["Optimized methods for the targeted surveillance of extended-spectrum beta-lactamase-producing\n                    <i>Escherichia coli<\/i>\n                    in human stool"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8938-1628","authenticated-orcid":false,"given":"Sarah","family":"Gallichan","sequence":"first","affiliation":[{"name":"Department of Clinical Sciences, Liverpool School of Tropical Medicine","place":["Liverpool, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sally","family":"Forrest","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Liverpool School of Tropical Medicine","place":["Liverpool, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Esther","family":"Picton-Barlow","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Liverpool School of Tropical Medicine","place":["Liverpool, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claudia","family":"McKeown","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Liverpool School of Tropical Medicine","place":["Liverpool, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Moore","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Liverpool School of Tropical Medicine","place":["Liverpool, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4413-3756","authenticated-orcid":false,"given":"Eva","family":"Heinz","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Liverpool School of Tropical Medicine","place":["Liverpool, United Kingdom"]},{"name":"Department of Vector Biology, Liverpool School of Tropical Medicine","place":["Liverpool, United Kingdom"]},{"name":"Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde","place":["Glasgow, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas A.","family":"Feasey","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Liverpool School of Tropical Medicine","place":["Liverpool, United Kingdom"]},{"name":"The School of Medicine, University of St Andrews","place":["St Andrews, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph M.","family":"Lewis","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Liverpool School of Tropical Medicine","place":["Liverpool, United Kingdom"]},{"name":"Department of Clinical Infection, Microbiology and Immunology, University of Liverpool","place":["Liverpool, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1132-5348","authenticated-orcid":false,"given":"Fabrice E.","family":"Graf","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Liverpool School of Tropical Medicine","place":["Liverpool, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_5_2_2","unstructured":"WHO publishes list of bacteria for which new antibiotics are urgently needed. Available from: https:\/\/www.who.int\/news\/item\/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed"},{"key":"e_1_3_5_3_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkm318"},{"key":"e_1_3_5_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jinf.2007.04.007"},{"key":"e_1_3_5_5_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12879-016-1430-5"},{"key":"e_1_3_5_6_2","first-page":"85","volume-title":"Escherichia coli: mechanisms of virulence","author":"Bettelheim KA","year":"1997","unstructured":"Bettelheim KA. 1997. Escherichia coli in the normal flora of humans and animals, p 85\u2013109. In Sussman M (ed), Escherichia coli: mechanisms of virulence. Cambridge University Press, Cambridge."},{"key":"e_1_3_5_7_2","doi-asserted-by":"publisher","DOI":"10.1093\/femsre\/fuac008"},{"key":"e_1_3_5_8_2","first-page":"68","volume-title":"Population genetics of bacteria: a tribute to Thomas S. Whittam","author":"Alm EW","year":"2011","unstructured":"Alm EW, Walk ST, Gordon DM. 2011. The niche of Escherichia coli, p 68\u201389. In Walk ST, Feng PCH (ed), Population genetics of bacteria: a tribute to Thomas S. Whittam. ASM Press, Washington, DC, USA."},{"key":"e_1_3_5_9_2","doi-asserted-by":"publisher","DOI":"10.1099\/jmm.0.027037-0"},{"key":"e_1_3_5_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tim.2018.08.004"},{"key":"e_1_3_5_11_2","unstructured":"Public Health England. 2016. UK standards for microbiology investigations detection of Enterobacteriaceae producing extended spectrum \u03b2-lactamases-standards-for-microbiology-investigations-Smi-quality-and-consistency-in-clinical-laboratories"},{"key":"e_1_3_5_12_2","unstructured":"World Health Organization & Advisory Group on Integrated Surveillance on AMR. 2021. WHO integrated global surveillance on ESBL-producing E. coli using a \u201cone health\u201d approach: implementation and opportunities. Available from: https:\/\/www.who.int\/publications\/i\/item\/9789240021402"},{"key":"e_1_3_5_13_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.14.4.933-951.2001"},{"key":"e_1_3_5_14_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dks340"},{"key":"e_1_3_5_15_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0075392"},{"key":"e_1_3_5_16_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1322678111"},{"key":"e_1_3_5_17_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.01613-20"},{"key":"e_1_3_5_18_2","doi-asserted-by":"publisher","DOI":"10.1016\/S2666-5247(21)00117-8"},{"key":"e_1_3_5_19_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12859-016-1016-7"},{"key":"e_1_3_5_20_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-45173-4"},{"key":"e_1_3_5_21_2","first-page":"117","article-title":"Heat treatment of bacteria: a simple method of DNA extraction for molecular techniques","volume":"41","author":"Dashti AA","year":"2009","unstructured":"Dashti AA, Jadaon MM, Abdulsamad AM, Dashti HM. 2009. Heat treatment of bacteria: a simple method of DNA extraction for molecular techniques. Kuwait Med J 41:117\u2013122.","journal-title":"Kuwait Med J"},{"key":"e_1_3_5_22_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btu170"},{"key":"e_1_3_5_23_2","unstructured":"Andrews S. 2010. FastQC: a quality control tool for high throughput sequence data. Available from: https:\/\/www.bioinformatics.babraham.ac.uk\/projects\/fastqc\/"},{"key":"e_1_3_5_24_2","doi-asserted-by":"publisher","DOI":"10.1099\/mgen.0.000131"},{"key":"e_1_3_5_25_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac920"},{"key":"e_1_3_5_26_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2006.05172.x"},{"key":"e_1_3_5_27_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gku1196"},{"key":"e_1_3_5_28_2","unstructured":"R Core Team. 2023. R: a language and environment for statistical computing. R Foundation for Statistical Computing Vienna Austria. https:\/\/www.R-project.org\/."},{"key":"e_1_3_5_29_2","unstructured":"Promega. DNA purification | DNA extraction methods. Available from: https:\/\/www.promega.co.uk\/resources\/guides\/nucleic-acid-analysis\/dna-purification\/"},{"key":"e_1_3_5_30_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2298"},{"key":"e_1_3_5_31_2","doi-asserted-by":"publisher","DOI":"10.1038\/nature11234"},{"key":"e_1_3_5_32_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.00378-15"},{"key":"e_1_3_5_33_2","doi-asserted-by":"publisher","DOI":"10.7717\/peerj.10572"},{"key":"e_1_3_5_34_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-44285-w"},{"key":"e_1_3_5_35_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1469-0691.2012.03999.x"},{"key":"e_1_3_5_36_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pcbi.1006496"},{"key":"e_1_3_5_37_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0222831"},{"key":"e_1_3_5_38_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.diagmicrobio.2020.115079"},{"key":"e_1_3_5_39_2","doi-asserted-by":"publisher","DOI":"10.1007\/s10096-021-04250-1"},{"key":"e_1_3_5_40_2","doi-asserted-by":"publisher","DOI":"10.1139\/facets-2016-0007"},{"key":"e_1_3_5_41_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.01039-19"},{"key":"e_1_3_5_42_2","unstructured":"Chromagar. CHROMagar ESBL. Available from: https:\/\/www.chromagar.com\/en\/product\/chromagar-esbl"},{"key":"e_1_3_5_43_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0160203"},{"key":"e_1_3_5_44_2","doi-asserted-by":"publisher","DOI":"10.1099\/jmm.0.47625-0"},{"key":"e_1_3_5_45_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1472-765X.1992.tb00782.x"},{"key":"e_1_3_5_46_2","volume-title":"Molecular detection of food borne pathogens","author":"Shah DH","year":"2010","unstructured":"Shah DH. 2010. Molecular detection of food borne pathogens. Taylor & Francis Group, CRC Press, USA."},{"key":"e_1_3_5_47_2","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-12-416999-9.00007-1"}],"container-title":["Microbiology Spectrum"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.01058-24","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.01058-24","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,1,7]],"date-time":"2025-01-07T09:12:16Z","timestamp":1736241136000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/spectrum.01058-24"}},"subtitle":[],"editor":[{"given":"Deena R.","family":"Altman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,1,7]]},"references-count":46,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2025,1,7]]}},"alternative-id":["10.1128\/spectrum.01058-24"],"URL":"https:\/\/doi.org\/10.1128\/spectrum.01058-24","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2024.04.02.24305201","asserted-by":"object"}]},"ISSN":["2165-0497"],"issn-type":[{"value":"2165-0497","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1,7]]},"assertion":[{"value":"2024-04-30","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2024-10-30","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2024-11-22","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e01058-24"}}