{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:14:05Z","timestamp":1785420845421,"version":"3.56.0"},"reference-count":23,"publisher":"American Society for Microbiology","issue":"12","license":[{"start":{"date-parts":[[2025,12,2]],"date-time":"2025-12-02T00:00:00Z","timestamp":1764633600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,12,2]],"date-time":"2025-12-02T00:00:00Z","timestamp":1764633600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2025,12,2]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      \u03b2-Lactamases are the leading mechanism of resistance to \u03b2-lactam antibiotics in Enterobacterales. Here, we report the detection and characteristics of SED-2, a new member of the\n                      <jats:italic toggle=\"yes\">Citrobacter sedlakii<\/jats:italic>\n                      class A chromosomal \u03b2-lactamase SED family. A\n                      <jats:italic toggle=\"yes\">C. sedlakii<\/jats:italic>\n                      isolate recovered during routine screening was subjected to whole genome sequencing. A new variant of SED-1 was identified and named SED-2.\n                      <jats:italic toggle=\"yes\">bla<\/jats:italic>\n                      SED-1 and\n                      <jats:italic toggle=\"yes\">bla<\/jats:italic>\n                      SED-2 were cloned into\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      DH10\u03b2. The donor\n                      <jats:italic toggle=\"yes\">C. sedlakii<\/jats:italic>\n                      and the recombinant\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      -SED-1 and\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      -SED-2 were further characterized. Antibiotic susceptibilities were determined using Sensititer. Kinetic assays were performed on purified SED-1 and SED-2. AlphaFold2 was used to predict the structure of SED-2 based on the primary amino acid sequence.\n                      <jats:italic toggle=\"yes\">bla<\/jats:italic>\n                      SED-2 differed from\n                      <jats:italic toggle=\"yes\">bla<\/jats:italic>\n                      SED-1 by six nucleotides, leading to one amino acid substitution (Q274R). Antibiotic susceptibility testing revealed that\n                      <jats:italic toggle=\"yes\">bla<\/jats:italic>\n                      SED-1 and\n                      <jats:italic toggle=\"yes\">bla<\/jats:italic>\n                      SED-2 conferred resistance to first-, second-, and third-generation cephalosporins but not to carbapenems. Kinetic studies of purified enzymes showed that SED-1 and SED-2 hydrolyzed ampicillin and cephalosporins but not meropenem. In comparison to SED-1, SED-2 had higher affinity and increased catalytic activity against the third-generation cephalosporins, especially ceftazidime. Moreover, compared with SED-1, SED-2 had a higher IC50 for clavulanic acid. 3D modeling of SED-2 predicted that the single amino acid substitution affected inhibitor-binding residues and led to a shift that destabilizes the enzyme active site, likely explaining the extended spectrum of activity. Our results highlight the diversity of \u03b2-lactamases naturally occurring in\n                      <jats:italic toggle=\"yes\">Citrobacter<\/jats:italic>\n                      spp. We demonstrate here a rare example of a single amino acid substitution that simultaneously expanded the spectrum of \u03b2-lactamase activity by increasing affinity towards ceftazidime and reduced susceptibility to the \u03b2-lactamase inhibitor clavulanic acid.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        In this work, we identified and characterized SED-2, a new member of the\n                        <jats:italic toggle=\"yes\">Citrobacter sedlakii<\/jats:italic>\n                        class A chromosomal \u03b2-lactamase SED family. While only differing from the SED-1 by a single amino acid, the affinity of SED-2 towards the third-generation cephalosporin ceftazidime was dramatically increased. At the same time, its susceptibility to the \u03b2-lactamase inhibitor clavulanic acid was reduced. This is a rare example of a single amino acid substitution, occurring outside of the active site, simultaneously affecting two distinct enzyme functions.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00224-25","type":"journal-article","created":{"date-parts":[[2025,10,27]],"date-time":"2025-10-27T12:00:33Z","timestamp":1761566433000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["A single amino acid substitution in SED-2 \u03b2-lactamase leads to increased inhibitor resistance and an extended substrate spectrum"],"prefix":"10.1128","volume":"13","author":[{"given":"S.","family":"Laviad-Shitrit","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/016n0q862","id-type":"ROR","asserted-by":"publisher"}],"name":"National Institute for Antibiotic Resistance and Infection Control, Israel Ministry of 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