A Novel Extended-Spectrum TEM-Type β-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 ␤-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 bla TEM-1 by three mutations leading to the following amino acid substitutions: Glu 104 3Lys, Met 182 3Thr, and Gly 238 3Ser (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 M) was 10-fold higher than that of TEM-3 (K i , 1.9 M). 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.Klebsiella pneumoniae is an important pathogen that is usually susceptible to extended-spectrum cephalosporins. However, strains producing extended-spectrum ␤-lactamases (ESBLs) were described in the early 1980s, and, since that time, there has been an increase in the incidence of ceftazidime (CAZ)resistant Klebsiella strains responsible for nosocomial outbreaks (8,16,27). In most cases, ESBLs are plasmid-encoded enzymes providing resistance to oxyiminocephalosporins (CAZ, cefotaxime [CTX], ceftriaxone, cefpirome, and cefepime [FEP]) and to aztreonam (ATM) (16,24). Cephamycins (cefoxitin [FOX] and cefotetan [CTT]), moxalactam (MOX), and carbapenems are stable toward most ESBLs, and strains producing such enzymes remained susceptible to these molecules (7,24). The molecular basis of the extended spectrum often involves point mutations within plasmid-mediated ␤-lactamase genes resulting in either single or multiple amino acid substitutions in the corresponding enzymes (7,16,24). We describe here a novel TEM-type ESBL able to hydrolyze MOX. The enzyme is produced by a clinical isolate of K. pneumoniae.(Part of this work was presented at the 37th Interscience

The genetic code constrains yet facilitates Darwinian evolution

“…1-bp substitutions). For example, TEM-52 differs from TEM-1 by three point mutations resulting in the E104K/M182T/G238S mutations (21) that increase cefotaxime resistance ∼4000-fold (16). The in vitro evolution of TEM-1 mimics its natural evolution (22).…”

Resistance to Oxyiminocephalosporins Mediated byblaTEM-52Genes in Salmonella Typhimurium from Humans in England and Wales

“…TEM-52 was described for the first time in 1998 in a Klebsiella isolate from France (Poyart et al, 1998). Since then it has been reported in various members of the Enterobacteriaceae family from several European countries (Italy, France, Hungary, Spain, Portugal) (De Champs et al, 2000;Vahaboglu et al, 2001;Perilli et al, 2002;Costa et al, 2004;Valverde et al, 2004), as well as in Korea (Jeong et al, 2004;Kim et al, 2004) and Canada (Mulvey et al, 2004).…”

Persistence of uropathogenic Escherichia coli strains in the host for long periods of time: relationship between phylogenetic groups and virulence factors

“…However, the enzyme TEM-52 differs from ancestral TEM-1 by three amino acid substitutions, of which Met 182 →Thr and Gly 238 →Ser enlarge the active site and give the enzyme higher affinities to cefotaxime. These mutations account for not only resistance to ESBLs, but also lower the susceptibility to β-lactamase inhibitors [22]. ESBLs are acquired β-lactamases that are encoded mainly by genes located on plasmids.…”