{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:36:57Z","timestamp":1760060217019,"version":"build-2065373602"},"reference-count":46,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2025,8,12]],"date-time":"2025-08-12T00:00:00Z","timestamp":1754956800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"JSPS KAKENHI","award":["22K08606"],"award-info":[{"award-number":["22K08606"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>Background: The Citrobacter genus harbors class C (AmpC) and class A \u03b2-lactamases. Citrobacter freundii produces an inducible AmpC \u03b2-lactamase controlled by the LysR-type transcriptional regulator AmpR and cytosolic amidase AmpD. Citrobacter sedlakii produces the class A \u03b2-lactamase Sed-1, whose expression is believed to be regulated by the transcriptional regulator SedR and AmpD. Objectives:C. sedlakii NR2807, isolated in Japan, is resistant to third-generation cephalosporins and displays extended-spectrum \u03b2-lactamase characteristics. Here, we sought to understand the mechanism for successful resistance to third-generation cephalosporins by investigating the regulators controlling Sed-1 production. Methods: Plasmids containing blaSed-1 and sedR (pCR2807) or truncated sedR (pCR2807\u0394SedR) were constructed and introduced into Escherichia coli. Antibiotic-resistant mutants of NR2807 were obtained, and enzyme kinetics were assessed. Results: The AmpD mutant (pCR2807\/ML4953) showed an 8-fold increase in cefotaxime MIC and an 8.46-fold increase in Sed-1 activity compared to the wild-type (pCR2807\/ML4947). However, induction of pCR2807\/ML4947 also led to a 1.32-fold higher Sed-1 activity, indicating semi-inducibility. Deletion of sedR (pCR2807\u0394SedR\/ML4947) led to a 4-fold decrease in cefotaxime MIC and 1.93-fold lower Sed-1 activity, confirming SedR as an activator. While wild-type C. sedlakii ATCC51115 is susceptible to third-generation cephalosporins, the AmpD mutation in NR2807 led to Sed-1 overproduction and resistance to this class of antibiotics. Finally, mutagenesis revealed that amino acid substitution in Sed-1 conferred resistance to ceftazidime and extended-spectrum \u03b2-lactamase characteristics. Conclusions: Sed-1 producers, though usually susceptible to third-generation cephalosporins, may develop extended-spectrum \u03b2-lactamase traits due to AmpD or Sed-1 mutations, thereby requiring careful monitoring.<\/jats:p>","DOI":"10.3390\/antibiotics14080823","type":"journal-article","created":{"date-parts":[[2025,8,13]],"date-time":"2025-08-13T07:45:31Z","timestamp":1755071131000},"page":"823","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Unique Regulation of Sed-1 \u03b2-Lactamase in Citrobacter sedlakii: Insights on Resistance to Third-Generation Cephalosporin"],"prefix":"10.3390","volume":"14","author":[{"given":"Mako","family":"Watanabe","sequence":"first","affiliation":[{"name":"Department of Microbiology and Infectious Diseases, Nara Medical University, 840 Shijo-cho, Kashihara 6348521, Nara, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2086-2591","authenticated-orcid":false,"given":"Ryuichi","family":"Nakano","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Infectious Diseases, Nara Medical University, 840 Shijo-cho, Kashihara 6348521, Nara, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Keizo","family":"Yamamoto","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Nara Medical University, 88 Shijo-cho, Kashihara 6340813, Nara, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Akiyo","family":"Nakano","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Infectious Diseases, Nara Medical University, 840 Shijo-cho, Kashihara 6348521, Nara, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuki","family":"Suzuki","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Infectious Diseases, Nara Medical University, 840 Shijo-cho, Kashihara 6348521, Nara, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kai","family":"Saito","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Infectious Diseases, Nara Medical University, 840 Shijo-cho, Kashihara 6348521, Nara, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Satoko","family":"Nakashima","sequence":"additional","affiliation":[{"name":"Division of Central Clinical Laboratory, Iwate Medical University Hospital, 1-1-1 Idaidori, Yahaba-cho, Shiwa-gun 0283694, Iwate, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kentaro","family":"Endo","sequence":"additional","affiliation":[{"name":"Division of Central Clinical Laboratory, Iwate Medical University Hospital, 1-1-1 Idaidori, Yahaba-cho, Shiwa-gun 0283694, Iwate, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kazuya","family":"Narita","sequence":"additional","affiliation":[{"name":"Division of Central Clinical Laboratory, Iwate Medical University Hospital, 1-1-1 Idaidori, Yahaba-cho, Shiwa-gun 0283694, Iwate, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hisakazu","family":"Yano","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Infectious Diseases, Nara Medical University, 840 Shijo-cho, Kashihara 6348521, Nara, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1016\/S1473-3099(17)30753-3","article-title":"Discovery, Research, and Development of New Antibiotics: The WHO Priority List of Antibiotic-Resistant Bacteria and Tuberculosis","volume":"18","author":"Tacconelli","year":"2018","journal-title":"Lancet Infect Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"100992","DOI":"10.1016\/j.lanmic.2024.100992","article-title":"Addressing Urgent Priorities in Antibiotic Development: Insights from WHO 2023 Antibacterial Clinical Pipeline Analyses","volume":"6","author":"Melchiorri","year":"2025","journal-title":"Lancet Microbe"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Husna, A., Rahman, M.M., Badruzzaman, A.T.M., Sikder, M.H., Islam, M.R., Rahman, M.T., Alam, J., and Ashour, H.M. 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