A single amino acid substitution in SED-2 β-lactamase leads to increased inhibitor resistance and an extended substrate spectrum

β-Lactamases are the leading mechanism of resistance to β-lactam antibiotics in Enterobacterales. Here, we report the detection and characteristics of SED-2, a new member of the <i>Citrobacter sedlakii</i> class A chromosomal β-lactamase SED family. A <i>C. sedlakii</i> isolate recovered during routine screening was subjected to whole genome sequencing. A new variant of SED-1 was identified and named SED-2. <i>bla</i>SED-1 and <i>bla</i>SED-2 were cloned into <i>E. coli</i> DH10β. The donor <i>C. sedlakii</i> and the recombinant <i>Escherichia coli</i>-SED-1 and <i>E. coli</i>-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. <i>bla</i>SED-2 differed from <i>bla</i>SED-1 by six nucleotides, leading to one amino acid substitution (Q274R). Antibiotic susceptibility testing revealed that <i>bla</i>SED-1 and <i>bla</i>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 β-lactamases naturally occurring in <i>Citrobacter</i> spp. We demonstrate here a rare example of a single amino acid substitution that simultaneously expanded the spectrum of β-lactamase activity by increasing affinity towards ceftazidime and reduced susceptibility to the β-lactamase inhibitor clavulanic acid.IMPORTANCEIn this work, we identified and characterized SED-2, a new member of the <i>Citrobacter sedlakii</i> class A chromosomal β-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 β-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.