A novel extended-spectrum TEM-type beta-lactamase (TEM-52) associated with decreased susceptibility to moxalactam in Klebsiella pneumoniae

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 beta-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 the blaT gene of pNEC865 differed from blaTEM-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 the blaT gene carried by pNEC865 was therefore designated blaTEM-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 microM) was 10-fold higher than that of TEM-3 (Ki, 1.9 microM). 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.

FIG. 1

Detection of the production of an ESBL by the double-disk synergy test. The bacterial strains were wild-type strain K. pneumoniae NEM865 (A) and E. coli TG1 containing pBGS18Ωbla_TEM-52 (B). Disks: 1, AMX-CA (AMX at 20 μg and CA at 10 μg); 2, CTX (30 μg); 3, MOX (40 μg); 4, ATM (30 μg); 5, CAZ (30 μg). Note the potentiation of cephalosporins, ATM, and MOX by CA.

FIG. 2

Restriction and Southern analyses of pNEC865. Lanes 1 and 1′ and lanes 2 and 2′, PstI digestion of ColE1::Tn3 and pNEC865, respectively; lanes 3 and 3′ and lanes 4 and 4′, BamHI-PstI digestion of ColE1::Tn3 and pNEC865, respectively. Plasmid ColE1::Tn3 was used as a probe. DNA fragments internal to Tn3 are indicated by white arrows.