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An unusual strain, GMU8049, was not susceptible to any of the antibiotics tested, including the novel \u03b2-lactam\/\u03b2-lactamase inhibitor combination ceftazidime-avibactam. Whole-genome sequencing (WGS) revealed that GMU8049 harbors a circular chromosome belonging to the rare ST257 and an IncX3 resistance plasmid. Genomic analysis revealed the coexistence of two \u03b2-lactamase genes, including plasmid-mediated\n              <jats:italic>bla<\/jats:italic>\n              <jats:sub>NDM-1<\/jats:sub>\n              and chromosomal\n              <jats:italic>bla<\/jats:italic>\n              <jats:sub>CMY<\/jats:sub>\n              encoding a novel CMY variant, combined with an outer membrane porin deficiency, which may account for the extreme resistance to \u03b2-lactams. Conjugation experiment confirmed that the\n              <jats:italic>bla<\/jats:italic>\n              <jats:sub>NDM-1<\/jats:sub>\n              resistance gene located on pGMU8049 could be successfully transferred to\n              <jats:italic>Escherichia coli<\/jats:italic>\n              EC600. The novel CMY variant had an amino acid substitution at position 106 (N106S) compared to the closely related CMY-51. Additionally, a GMU8049-specific truncation in an OmpK37 variant that produces a premature stop codon. Moreover, a variety of chromosome-located efflux pump coding genes and virulence-related genes were also identified. Analysis of strain GMU8049 in the context of other\n              <jats:italic>C. freundii<\/jats:italic>\n              strains reveals an open pan-genome and the presence of mobile genetic elements that can mediate horizontal gene transfer of antimicrobial resistance and virulence genes. Our work provides comprehensive insights into the genetic mechanisms of highly resistant\n              <jats:italic>C. freundii<\/jats:italic>\n              , highlighting the importance of genomic surveillance of this opportunistic pathogen as a high-risk population for emerging resistance and pathogenicity.\n            <\/jats:p>\n            <jats:sec>\n              <jats:title>IMPORTANCE<\/jats:title>\n              <jats:p>\n                Emerging pathogens exhibiting multi-, extremely, and pan-drug resistance are a major concern for hospitalized patients and the healthcare community due to limited antimicrobial treatment options and the potential for spread. Genomic technologies have enabled clinical surveillance of emerging pathogens and modeling of the evolution and transmission of antimicrobial resistance and virulence. Here, we report the genomic characterization of an extremely drug-resistant ST257\n                <jats:italic>Citrobacter freundii<\/jats:italic>\n                clinical isolate. Genomic analysis of GMU8049 with a rare ST type and unusual phenotypes can provide information on how this extremely resistant clinical isolate has evolved, including the acquisition of\n                <jats:italic>bla<\/jats:italic>\n                <jats:sub>NDM-1<\/jats:sub>\n                via the IncX3 plasmid and accumulation through chromosomal mutations leading to a novel CMY variant and deficiency of the outer membrane porin OmpK37. Our work highlights that the emergence of extremely resistant\n                <jats:italic>C. freundii<\/jats:italic>\n                poses a significant challenge to the treatment of clinical infections. Therefore, great efforts must be made to specifically monitor this opportunistic pathogen.\n              <\/jats:p>\n            <\/jats:sec>\n          <\/jats:sec>","DOI":"10.1128\/spectrum.04254-23","type":"journal-article","created":{"date-parts":[[2024,10,10]],"date-time":"2024-10-10T13:07:49Z","timestamp":1728565669000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":11,"title":["Investigation of\n            <i>Citrobacter freundii<\/i>\n            clinical isolates in a Chinese hospital during 2020\u20132022 revealed genomic characterization of an extremely drug-resistant\n            <i>C. freundii<\/i>\n            ST257 clinical strain GMU8049 co-carrying\n            <i>bla<\/i>\n            <sub>NDM-1<\/sub>\n            and a novel\n            <i>bla<\/i>\n            <sub>CMY<\/sub>\n            variant"],"prefix":"10.1128","volume":"12","author":[{"given":"Mujie","family":"Zhang","sequence":"first","affiliation":[{"name":"Department of Clinical Laboratory, Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3355-3683","authenticated-orcid":true,"given":"Zhiqiu","family":"Yin","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Baozhu","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhanpeng","family":"Yu","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaxin","family":"Liang","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 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