{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T14:14:15Z","timestamp":1788272055478,"version":"build-2803163510"},"reference-count":27,"publisher":"American Society for Microbiology","issue":"4","license":[{"start":{"date-parts":[[2024,4,2]],"date-time":"2024-04-02T00:00:00Z","timestamp":1712016000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2024,4,2]],"date-time":"2024-04-02T00:00:00Z","timestamp":1712016000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001809","name":"MOST | National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["32172907"],"award-info":[{"award-number":["32172907"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001809","name":"MOST | National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["32373061"],"award-info":[{"award-number":["32373061"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"name":"JST | Jiangsu Provincial Key Research and Development Program","award":["BE2023332"],"award-info":[{"award-number":["BE2023332"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2024,4,2]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Tigecycline and colistin were referred to as the \u201clast resort\u201d antibiotics in defending against carbapenem-resistant, Gram-negative bacterial infections, and are currently widely used in clinical treatment. However, the emergence and prevalence of plasmid-mediated\n                      <jats:italic>tet<\/jats:italic>\n                      (X4) and\n                      <jats:italic>mcr-1<\/jats:italic>\n                      genes pose a serious threat to the therapeutic application of tigecycline and colistin, respectively. In this research, a tigecycline- and colistin-resistant bacteria resensitization system was developed based on efficient and specific DNA damage caused by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Associated Protein 9 (Cas9) nucleases. A conjugation method was used to deliver the resensitization system, which harbors two single-guide RNAs targeting\n                      <jats:italic>tet<\/jats:italic>\n                      (X4) and\n                      <jats:italic>mcr-1<\/jats:italic>\n                      genes and constitutively expressed Cas9. The conjugation efficiency was nearly 100% after conjugation condition optimization\n                      <jats:italic>in vitro<\/jats:italic>\n                      , and the resensitivity efficiency for clinical isolates was over 90%. In addition, when performing resensitization\n                      <jats:italic>in vivo<\/jats:italic>\n                      , the resistance marker was replaced with a glutamate-based, chromosomal, plasmid-balanced lethal system to prevent the introduction of additional resistance genes in clinical settings, making this strategy a therapeutic approach to combat the\n                      <jats:italic>in vivo<\/jats:italic>\n                      spread of antibiotic resistance genes (ARGs) among bacterial pathogens. As a proof of concept, this resensitive system can significantly decrease the counts of tigecycline- and colistin-resistant bacteria to 1%\n                      <jats:italic>in vivo<\/jats:italic>\n                      . Our study demonstrates the efficacy and adaptability of CRISPR-Cas systems as powerful and programmable antimicrobials in resensitizing\n                      <jats:italic>tet<\/jats:italic>\n                      (X4)- and\n                      <jats:italic>mcr-1-<\/jats:italic>\n                      mediated, tigecycline- and colistin-resistant strains, and opens up new pathways for the development of CRISPR-based tools for selective bacterial pathogen elimination and precise microbiome composition change.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      The emergence of plasmid-encoded\n                      <jats:italic>tet<\/jats:italic>\n                      (X4) and\n                      <jats:italic>mcr-1<\/jats:italic>\n                      isolated from human and animal sources has affected the treatment of tigecycline and colistin, and has posed a significant threat to public health. Tigecycline and colistin are considered as the \u201clast line of defense\u201d for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, so there is an urgent need to find a method that can resensitize\n                      <jats:italic>tet<\/jats:italic>\n                      (X4)-mediated tigecycline-resistant and\n                      <jats:italic>mcr-1<\/jats:italic>\n                      -mediated colistin-resistant bacteria. In this study, we developed a glutamate-based, chromosomal, plasmid-balanced lethal conjugative CRISPR\/Cas9 system, which can simultaneously resensitize\n                      <jats:italic>tet<\/jats:italic>\n                      (X4)-mediated tigecycline-resistant and\n                      <jats:italic>mcr-1<\/jats:italic>\n                      -mediated colistin-resistant\n                      <jats:italic>Escherichia coli<\/jats:italic>\n                      . The counts of tigecycline- and colistin-resistant bacteria decreased to 1%\n                      <jats:italic>in vivo<\/jats:italic>\n                      after the resensitization system was administered. This study opens up new pathways for the development of CRISPR-based tools for selective bacterial pathogen elimination and precise microbiome composition change.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.03884-23","type":"journal-article","created":{"date-parts":[[2024,2,22]],"date-time":"2024-02-22T09:01:05Z","timestamp":1708592465000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":21,"title":["Resensitizing tigecycline- and colistin-resistant\n                    <i>Escherichia coli<\/i>\n                    using an engineered conjugative CRISPR\/Cas9 system"],"prefix":"10.1128","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3473-0186","authenticated-orcid":false,"given":"Haijie","family":"Zhang","sequence":"first","affiliation":[{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, China"},{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bo","family":"Chen","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zeyu","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kai","family":"Peng","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9622-6471","authenticated-orcid":true,"given":"Yuan","family":"Liu","sequence":"additional","affiliation":[{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, China"},{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou, China"},{"name":"Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China"},{"name":"Institute of Comparative Medicine, Yangzhou University, Yangzhou, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0593-2475","authenticated-orcid":true,"given":"Zhiqiang","family":"Wang","sequence":"additional","affiliation":[{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, China"},{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou, China"},{"name":"Institute of Comparative Medicine, Yangzhou University, Yangzhou, 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DNA vaccines: Safety aspect assessment and regulation. In Developmental Biology (Basel). 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