A Novel Extended-Spectrum TEM-Type β-Lactamase (TEM-52) Associated with Decreased Susceptibility to Moxalactam in Klebsiella pneumoniae

Abstract

ABSTRACT Klebsiella pneumoniae NEM865 was isolated from the culture of a stool sample from a patient previously treated with ceftazidime (CAZ). Analysis of this strain by the disk diffusion test revealed synergies between amoxicillin-clavulanate (AMX-CA) and CAZ, AMX-CA and cefotaxime (CTX), AMX-CA and aztreonam (ATM), and more surprisingly, AMX-CA and moxalactam (MOX). Clavulanic acid (CA) decreased the MICs of CAZ, CTX, and MOX, which suggested that NEM865 produced a novel extended-spectrum β-lactamase. Genetic, restriction endonuclease, and Southern blot analyses revealed that the resistance phenotype was due to the presence in NEM865 of a 13.5-kb mobilizable plasmid, designated pNEC865, harboring a Tn 3 -like element. Sequence analysis revealed that the blaT gene of pNEC865 differed from bla TEM-1 by three mutations leading to the following amino acid substitutions: Glu 104 →Lys, Met 182 →Thr, and Gly 238 →Ser (Ambler numbering). The association of these three mutations has thus far never been described, and the blaT gene carried by pNEC865 was therefore designated bla TEM-52 . The enzymatic parameters of TEM-52 and TEM-3 were found to be very similar except for those for MOX, for which the affinity of TEM-52 ( K i , 0.16 μM) was 10-fold higher than that of TEM-3 ( K i , 1.9 μM). Allelic replacement analysis revealed that the combination of Lys 104 , Thr 182 , and Ser 238 was responsible for the increase in the MICs of MOX for the TEM-52 producers.