Novel Class A β-Lactamase Sed-1 from Citrobacter sedlakii : Genetic Diversity of β-Lactamases within the Citrobacter Genus

Abstract

ABSTRACT Citrobacter sedlakii 2596, 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, bla Sed-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%), and Proteus vulgaris (63%) and 71% identity with the plasmid-mediated enzyme MEN-1. A gene coding for a LysR transcriptional regulator was found upstream from bla Sed-1 . This regulator, named SedR, displayed 90% identity with the AmpR sequence of the chromosomal β-lactamase from C. koseri and 63 and 50% identity with the AmpR sequences of P. vulgaris and Enterobacter cloacae , respectively. By using DNA-DNA hybridization, a bla Sed-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. koseri studied. Two DNA fragments were amplified and sequenced from the reference strains of C. sedlakii CIP-105037 and C. rodentium CIP-104675 using two primers specific for bla Sed-1 . They shared 98 and 80% identity with bla Sed-1 , respectively, confirming the diversity of the chromosomally encoded class A β-lactamases found in Citrobacter .