Characterization of the Novel CMT Enzyme TEM-154

Abstract

TEM-154, identified in Portugal in 2004, associated the substitutions observed in the extended-spectrum ␤-lactamase (ESBL) TEM-12 and in the inhibitor-resistant penicillinase (IRT) TEM-33. This enzyme exhibited hydrolytic activity against ceftazidime and a low level of resistance to clavulanic acid. Surprisingly, the substitution Met69Leu enhanced the catalytic efficiency of oxyimino ␤-lactams conferred by the substitution Arg164Ser. Its discovery confirms the dissemination of the complex mutant group of TEM enzymes in European countries.TEM-type ␤-lactamases are the most prevalent mechanism of resistance to ␤-lactam antibiotics in Enterobacteriaceae. Among these ␤-lactamases, extended spectrum ␤-lactamases (ESBL) and inhibitor-resistant penicillinases (IRT) have emerged from the parental penicillinases TEM-1 and TEM-2 (2). Since the 1990s, another subgroup of enzymes that combine the substitutions observed in the ESBL and in the IRT appeared; its members were designated complex mutant TEM (CMT) (21). Nine different CMT, mainly in E. coli strains, have been described: TEM-50, TEM-68, TEM-89, TEM-121, TEM-109, TEM-125, TEM-151, TEM-152, and TEM-158, most of which have been identified in France (7,14,(16)(17)(18)(19)(20)(21).In 2004, during a 2-year study of ESBL-producing Enterobacteriaceae from different Portuguese clinical settings (12), Machado et al. identified a novel TEM enzyme that associated the Arg164Ser substitution observed in the ESBL TEM-12 (4) with the Met69Leu substitution observed in TEM-33 (IRT-5) (3). This ␤-lactamase, designated TEM-154, was produced by an Escherichia coli strain, H258, which is resistant to penicillins (alone or associated with clavulanic acid), ceftazidime, all tested aminoglycosides except amikacin, all tested quinolones, sulfamethoxazole, tetracycline, and chloramphenicol.In this study, we characterized the genetic support and the enzymatic activity of TEM-154. E. coli H258 produces only one ␤-lactamase, which has a pI of 5.3. The French double-disk synergy test and CLSI MIC testing confirmed the production of an ESBL by this strain (5, 6). The TEM-154-harboring plasmid did not transfer in mating experiments (19). However, an E. coli DH5␣ transformant harboring the parental phenotype of resistance to ␤-lactams was obtained throughout electroporation of plasmid DNA. Plasmid DNAs were extracted from the clinical strain and transformant by the method of Kado and Liu (8). Plasmid size was determined by comparison with those of plasmids Rsa (39 kb), TP114 (61 kb), pCFF04 (85 kb), and pCFF14 (180 kb), as previously described (15). Plasmid content analysis revealed one plasmid of about 180 kb. TEM-specific PCR and sequencing experiments were performed on the clinical strain H258 and on the transformant as previously described (17) and confirmed the presence of bla . This gene harbored a pattern of silent mutations identical to those of bla TEM-1a and a promoting sequence, P a /P b (11).E. coli DH5␣ clones producing TEM-154, TEM-12, TEM-33, and TEM-1 were obtained using the vector...

In Vivo Evolution of CTX-M-215, a Novel Narrow-Spectrum β-Lactamase in an Escherichia coli Clinical Isolate Conferring Resistance to Mecillinam

“…The purities of CTX-M proteins were more than 95% as estimated by SDS-PAGE. Hydrolysis of ␤-lactams by CTX-M enzymes was measured using a UV-2700 spectrophotometer (Shimadzu, Kyoto, Japan) (2,12). The overall catalytic efficiency (k cat /K m ) of CTX-M-14 and CTX-M-125 was similar to that in a previous report (Table 2) (13).…”

Detection of the mcr-1 Gene in a Multidrug-Resistant Escherichia coli Isolate from an Austrian Patient

“…The bla TEM-154 -containing contig (8,459 bp) matched E. coli plasmid R1 transposon Tn4 (GenBank accession number HM749966.1) to 99% (3 mismatches) (11). The presence of this complex mutant TEM-type ESBL has not yet been associated with mcr-1 carriage (2,12). To assess the classic multilocus sequence type (MLST), ST10 was extracted in silico from WGS data using the Warwick MLST scheme.…”

CTX-M-190, a Novel β-Lactamase Resistant to Tazobactam and Sulbactam, Identified in an Escherichia coli Clinical Isolate

“…The hydrolysis of ␤-lactam compounds by CTX-M-190 and CTX-M-55 was monitored by absorbance variation at the appropriate wavelengths in phosphatebuffered saline (137 mM NaCl, 2.7 mM KCl, 1.8 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 ) at 30°C using a UV-2700 spectrophotometer (Shimadzu, Kyoto, Japan), and steady-state kinetic parameters (K m , k cat , and k cat /K m ) were determined as previously described (9,10). As shown by the results in Table 3, the highest catalytic efficiency of CTX-M-190 was observed toward ampicillin and cefotaxime, with k cat /K m ratios of 2.2 and 14.5 M Ϫ1 s Ϫ1 , respectively.…”

“…The above-mentioned BamHI-EcoRI-digested PCR products were ligated into a BamHIEcoRI-digested pET28a(ϩ) expression vector, which was then introduced by electroporation into E. coli BL21(DE3) (Novagen, Darmstadt, Germany). The expression of the three kinds of CTX-M ␤-lactamases was induced by 0.75 mM IPTG (isopropyl-␤-Dthiogalactopyranoside) as previously described (9), and proteins were purified by using nickel magnetic beads for His tag protein purification (Biotool, Houston, TX, USA), following the manufacturer's instructions. Purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.…”