Unique Regulation of Sed-1 β-Lactamase in Citrobacter sedlakii: Insights on Resistance to Third-Generation Cephalosporin

<b>Background:</b> The <i>Citrobacter</i> genus harbors class C (AmpC) and class A β-lactamases. <i>Citrobacter freundii</i> produces an inducible AmpC β-lactamase controlled by the LysR-type transcriptional regulator AmpR and cytosolic amidase AmpD. <i>Citrobacter sedlakii</i> produces the class A β-lactamase Sed-1, whose expression is believed to be regulated by the transcriptional regulator SedR and AmpD. <b>Objectives:</b><i>C. sedlakii</i> NR2807, isolated in Japan, is resistant to third-generation cephalosporins and displays extended-spectrum β-lactamase characteristics. Here, we sought to understand the mechanism for successful resistance to third-generation cephalosporins by investigating the regulators controlling Sed-1 production. <b>Methods:</b> Plasmids containing <i>bla</i>_Sed-1 and <i>sedR</i> (pCR2807) or truncated <i>sedR</i> (pCR2807ΔSedR) were constructed and introduced into <i>Escherichia coli</i>. Antibiotic-resistant mutants of NR2807 were obtained, and enzyme kinetics were assessed. <b>Results:</b> 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 <i>sedR</i> (pCR2807ΔSedR/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 <i>C. sedlakii</i> 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 β-lactamase characteristics. <b>Conclusions:</b> Sed-1 producers, though usually susceptible to third-generation cephalosporins, may develop extended-spectrum β-lactamase traits due to AmpD or Sed-1 mutations, thereby requiring careful monitoring.