Novel Class A β-Lactamase Sed-1 fromCitrobacter sedlakii: Genetic Diversity of β-Lactamases within the CitrobacterGenus

ABSTRACTCitrobacter sedlakii2596, a clinical strain resistant to aminopenicillins, carboxypenicillins, and early cephalosporins such as cephalothin, but remaining susceptible to acylureidopenicillins, carbapenems, and later cephalosporins such as cefotaxime, was isolated from the bile of a patient treated with β-lactam and quinolone antibiotics. The isolate produced an inducible class A β-lactamase of pI 8.6, named Sed-1, which was purified. Characterized by a molecular mass of 30 kDa, Sed-1 preferentially hydrolyzed benzylpenicillin, cephalothin, and cloxacillin. The corresponding gene,blaSed-1, was cloned and sequenced. Its deduced amino acid sequence shared more than 60% identity with the chromosome-encoded β-lactamases from Citrobacter koseri(formerly C. diversus) (84%), Klebsiella oxytoca(74%), Serratia fonticola(67%), andProteus vulgaris(63%) and 71% identity with the plasmid-mediated enzyme MEN-1. A gene coding for a LysR transcriptional regulator was found upstream from blaSed-1. This regulator, named SedR, displayed 90% identity with the AmpR sequence of the chromosomal β-lactamase from C. koseriand 63 and 50% identity with the AmpR sequences of P. vulgarisand Enterobacter cloacae, respectively. By using DNA-DNA hybridization, a blaSed-1-like gene was identified in two reference strains, C. sedlakii(CIP-105037) and Citrobacter rodentium(CIP-104675), but not in the 18 strains of C. koseristudied. Two DNA fragments were amplified and sequenced from the reference strains ofC. sedlakiiCIP-105037 and C. rodentiumCIP-104675 using two primers specific forblaSed-1. They shared 98 and 80% identity withblaSed-1, respectively, confirming the diversity of the chromosomally encoded class A β-lactamases found inCitrobacter.