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

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 Tn3 -like element. Sequence analysis revealed that theblaT gene of pNEC865 differed frombla TEM-1 by three mutations leading to the following amino acid substitutions: Glu104 →Lys, Met182 →Thr, and Gly238 →Ser (Ambler numbering). The association of these three mutations has thus far never been described, and theblaT gene carried by pNEC865 was therefore designatedbla 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 (Ki , 0.16 μM) was 10-fold higher than that of TEM-3 (Ki , 1.9 μM). Allelic replacement analysis revealed that the combination of Lys104 , Thr182 , and Ser238 was responsible for the increase in the MICs of MOX for the TEM-52 producers.