{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,4]],"date-time":"2026-11-04T23:42:42Z","timestamp":1793835762669,"version":"4.6.1"},"reference-count":58,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2023,9,12]],"date-time":"2023-09-12T00:00:00Z","timestamp":1694476800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000865","name":"Bill and Melinda Gates Foundation","doi-asserted-by":"publisher","award":["OPP1138570"],"award-info":[{"award-number":["OPP1138570"]}],"id":[{"id":"10.13039\/100000865","id-type":"DOI","asserted-by":"publisher"}]},{"name":"USDA","award":["Hatch IDA01406"],"award-info":[{"award-number":["Hatch IDA01406"]}]},{"name":"USDA","award":["Hatch ID01574, IDA 01726"],"award-info":[{"award-number":["Hatch ID01574, IDA 01726"]}]},{"name":"NIH","award":["P20GM103408"],"award-info":[{"award-number":["P20GM103408"]}]},{"name":"USDA","award":["2022-67017-36315"],"award-info":[{"award-number":["2022-67017-36315"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>\n                    Pathogenic\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    and\n                    <jats:italic>Salmonella enterica<\/jats:italic>\n                    pose serious public health threats due to their ability to cause severe gastroenteritis and life-threatening sequela, particularly in young children. Moreover, the emergence and dissemination of antibiotic resistance in these bacteria have complicated control of infections. Alternative strategies that effectively target these enteric pathogens and negate or reduce the need of antibiotics are urgently needed. Such an alternative is the CRISPR-Cas9 system because it can generate sequence-specific lethal double stranded DNA breaks. In this study, two self-transmissible broad host range conjugative plasmids, pRK24 and pBP136, were engineered to deliver multiplexed CRSIPR-Cas9 systems that specifically target Enterohemorrhagic and Enteropathogenic strains of\n                    <jats:italic>E<\/jats:italic>\n                    .\n                    <jats:italic>coli<\/jats:italic>\n                    (EHEC and EPEC),\n                    <jats:italic>S<\/jats:italic>\n                    .\n                    <jats:italic>enterica<\/jats:italic>\n                    , and\n                    <jats:italic>bla<\/jats:italic>\n                    <jats:sub>CMY-2<\/jats:sub>\n                    antibiotic resistance plasmids. Using\n                    <jats:italic>in vitro<\/jats:italic>\n                    mating assays, we show that the conjugative delivery of pRK24-CRISPR-Cas9 carrying guide RNAs to the EPEC\/EHEC\n                    <jats:italic>eae<\/jats:italic>\n                    (intimin) gene can selectively kill enterohemorrhagic\n                    <jats:italic>E<\/jats:italic>\n                    .\n                    <jats:italic>coli<\/jats:italic>\n                    O157\n                    <jats:italic>eae<\/jats:italic>\n                    <jats:sup>+<\/jats:sup>\n                    cells (3 log kill at 6 h) but does not kill the isogenic\n                    <jats:italic>\u0394eae<\/jats:italic>\n                    mutant (P&lt;0.001). Similar results were also obtained with a pBP136 derivative, pTF16, carrying multiplexed guide RNAs targeting\n                    <jats:italic>E<\/jats:italic>\n                    .\n                    <jats:italic>coli eae<\/jats:italic>\n                    and the\n                    <jats:italic>S<\/jats:italic>\n                    .\n                    <jats:italic>enterica ssaN<\/jats:italic>\n                    gene coding for the type III secretion ATPase. Another pBP136 derivative, TF18, carries guide RNAs targeting\n                    <jats:italic>S<\/jats:italic>\n                    .\n                    <jats:italic>enterica ssaN<\/jats:italic>\n                    and the antibiotic resistance gene,\n                    <jats:italic>bla<\/jats:italic>\n                    <jats:sub>CMY-2<\/jats:sub>\n                    , carried on the multi-drug resistant pAR06302. Introduction of pTF18 into bacteria harboring pAR06302 showed plasmids were cured at an efficiency of 53% (P&lt;0.05). Using a murine neonate EPEC infection model, pTF16 was delivered by a murine derived\n                    <jats:italic>E<\/jats:italic>\n                    .\n                    <jats:italic>coli<\/jats:italic>\n                    strain to EPEC infected mice and showed significant reductions of intestinal EPEC (P&lt;0.05). These results suggest that establishing conjugative CRISPR-Cas9 antimicrobials in the intestinal microbiome may provide protection from enteric pathogens and reduce antibiotic resistance without disrupting the normal microbiota.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0291520","type":"journal-article","created":{"date-parts":[[2023,9,12]],"date-time":"2023-09-12T13:25:49Z","timestamp":1694525149000},"page":"e0291520","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":25,"title":["Engineering conjugative CRISPR-Cas9 systems for the targeted control of enteric pathogens and antibiotic resistance"],"prefix":"10.1371","volume":"18","author":[{"given":"Haiqing","family":"Sheng","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yansong","family":"Xue","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Allan B.","family":"Caplan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolyn J.","family":"Hovde","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3202-9049","authenticated-orcid":true,"given":"Scott A.","family":"Minnich","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2023,9,12]]},"reference":[{"issue":"1","key":"pone.0291520.ref001","doi-asserted-by":"crossref","first-page":"7","DOI":"10.3201\/eid1701.P11101","article-title":"Foodborne illness acquired in the United States\u2014major pathogens","volume":"17","author":"E Scallan","year":"2011","journal-title":"Emerg Infect Dis"},{"issue":"6","key":"pone.0291520.ref002","doi-asserted-by":"crossref","DOI":"10.1128\/microbiolspec.EHEC-0014-2013","article-title":"Public Health Microbiology of Shiga Toxin-Producing Escherichia coli.","volume":"2","author":"A Caprioli","year":"2014","journal-title":"Microb Spectr"},{"issue":"3","key":"pone.0291520.ref003","doi-asserted-by":"crossref","first-page":"407","DOI":"10.3201\/eid1903.111866","article-title":"Attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data, United States, 1998\u20132008","volume":"19","author":"JA Painter","year":"2013","journal-title":"Emerg Infect Dis"},{"issue":"7","key":"pone.0291520.ref004","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1179\/2047773215Y.0000000030","article-title":"Antimicrobial resistance: a global multifaceted phenomenon.","volume":"109","author":"F Prestinaci","year":"2015","journal-title":"Pathog Glob Health"},{"issue":"2","key":"pone.0291520.ref005","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1517\/14656566.2015.993381","article-title":"Antimicrobial resistance: impact on clinical and economic outcomes and the need for new antimicrobials.","volume":"16","author":"AK Thabit","year":"2015","journal-title":"Expert Opin Pharmacother"},{"issue":"14","key":"pone.0291520.ref006","doi-asserted-by":"crossref","first-page":"1447","DOI":"10.1001\/jama.2013.2824","article-title":"Genomes of microbes inhabiting the body offer clues to human health and disease","volume":"309","author":"MJ Friedrich","year":"2013","journal-title":"Jama"},{"issue":"1","key":"pone.0291520.ref007","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1053\/j.gastro.2008.10.080","article-title":"Microbes in gastrointestinal health and disease","volume":"136","author":"AS Neish","year":"2009","journal-title":"Gastroenterology"},{"issue":"5","key":"pone.0291520.ref008","first-page":"314","article-title":"Early Life Experience and Gut Microbiome: The Brain-Gut-Microbiota Signaling System. 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