E240V Substitution Increases Catalytic Efficiency toward Ceftazidime in a New Natural TEM-Type Extended-Spectrum β-Lactamase, TEM-149, from Enterobacter aerogenes and Serratia marcescens Clinical Isolates

Abstract

The aim of this study was to characterize a novel extended-spectrum ␤-lactamase that belongs to the TEM family, the TEM-149 enzyme, and that was isolated from the urine of two hospitalized patients from different hospitals in southern Italy. The peculiarity of this enzyme was the finding of a valine residue at position 240. The array of amino acid substitutions found in TEM-149 was as follows: E104K, R164S, M182T, and E240V. A reversion of a threonine residue at position 182 was also performed to create a new mutant, TEM-149 T182M , in order to assess the contribution of this substitution on the kinetic profile and the stability of TEM-149. The bla TEM-149 and bla TEM-149/T182M genes were cloned into pBC-SK, and the corresponding enzymes were purified from recombinant Escherichia coli HB101 by the same procedure. Both enzymes hydrolyzed all ␤-lactams tested, with a preference for ceftazidime, which was found to be the best substrate. By comparison of the kinetic parameters of the TEM-149 and the TEM-149 T182M enzymes, a reduction of the catalytic efficiency for the TEM-149 T182M mutant was observed against all substrates tested except benzylpenicillin, cefotaxime, and aztreonam. Tazobactam, clavulanic acid, and sulbactam were good inhibitors of the TEM-149 ␤-lactamase.Bacterial resistance to ␤-lactam antibiotics is primarily mediated by the production of ␤-lactamases, a group of enzymes which are able to catalyze the hydrolysis of the amide bond in the ␤-lactam ring. The rapid spread of ␤-lactam resistance among different bacterial species is facilitated by the transmission of ␤-lactamase-encoding genes via mobile genetic elements, such as plasmids, transposons, and integron-borne mobile gene cassettes (7,9).The massive use of expanded-spectrum cephalosporins since the 1980s has selected for the emergence of ␤-lactamases that can hydrolyze these compounds (the so-called extended-spectrum ␤-lactamases [ESBLs]) in the clinical setting. Since the first detection of plasmid-mediated ESBLs, the SHV-2 and TEM-3 enzymes (22), several ESBL types, and a large number of allelic variants have been described, mostly in the family Enterobacteriaceae but also in other gram-negative pathogens; and their dissemination represents a worldwide problem in hospitalized and community patients (25). Classical ESBLs have evolved from the broad-spectrum TEM-1, TEM-2, and SHV-type enzymes by amino acid substitutions (2, 3, 16). Today the number of known TEM-type and SHV-type ESBL variants isolated from clinical strains is very high and continues to grow each year, which is indicative of the ongoing evolution of these enzymes (G. Jacoby and K. Bush, http://www.lahey.org /studies/webt.htm). Recently, several types of non-TEM and non-SHV ESBLs (e.g., CTX-M, PER, VEB, GES, TLA, BES, and BEL) have also emerged in gram-negative bacteria (2, 16).The TEM-type variants remain among the most prevalent ESBLs (2, 16). They are derived from TEM-1 or TEM-2 enzymes by changes in the substrate specificity due to amino acid substitutions that occur at sp...

R164H and V240H Replacements by Site-Directed Mutagenesis of TEM-149 Extended-Spectrum β-Lactamase: Kinetic Analysis of TEM-149 H240 and TEM-149 H164-H240 Laboratory Mutants

“…In TEM-type enzymes, position 240 is generally taken up by a glutamate, which can interact with the amino group of the amino-thiazole substituent of cephalosporins. In wild-type TEM-149, this position is occupied by the nonpolar residue valine, which can facilitate the accommodation of the bulky oxyimino substituent of ceftazidime (6). This results in an increase in the affinity of the enzyme for the substrate.…”

“…During the last nationwide survey of ESBLs undertaken in Italy in 2003, a TEM-149 enzyme was isolated from Enterobacter aerogenes and Serratia marcescens (6). Compared to the TEM-1 sequence, TEM-149 showed an original array of amino acid changes: E104K, R164S, M182T, and E240V.…”

Non-phenotypic tests to detect and characterize antibiotic resistance mechanisms in Enterobacteriaceae

“… QRDR, quinolone resistance determining region; AMEs, aminoglycoside modifying enzymes. a Amino acidic substitutions E104K, R164S/H, G238S and E240K for TEM; D179N, G238S and E240K for SHV (Gniadkowski, 2008; Perilli, et al, 2008; Randegger, Keller, Irla, Wada, & Hachler, 2000); b Amino acidic substitution G170A/S (Girlich, Poirel, Szczepanowski, Schluter, & Nordmann, 2012); c Lead to modification of S83 and D87 (Jacoby, 2005; Qiang, et al, 2002); d Lead to modification of S80 and E84 (Qiang, et al, 2002) (Jacoby, 2005) …”

“… a Amino acidic substitutions E104K, R164S/H, G238S and E240K for TEM; D179N, G238S and E240K for SHV (Gniadkowski, 2008; Perilli, et al, 2008; Randegger, Keller, Irla, Wada, & Hachler, 2000); …”

Kinetic Study of the Effect of Histidines 240 and 164 on TEM-149 Enzyme Probed by β-Lactam Inhibitors

“…The values of k i (first-order rate constant which characterized the EC* accumulation) were obtained by time course hydrolysis of nitrocefin following equations 2 and 3, as previously determined (5, 6): (3) where v 0 , v t , and v ss are the rate transformations of the substrate at time zero, at time t, and at steady state: Thirteen compounds, including penicillin, penems, carbapenems, monobactams, carbacephem, and cephamycin, were tested as molecules able to inhibit or inactivate the enzymes TEM-149, TEM-149 H240 (single mutant), and TEM-149 H164-H240 (double mutant). Tables 1 and 2 summarize the values of K i , K, k ϩ2 , k ϩ3 , and k ϩ2 /K calculated for the three enzymes.…”