Jimena Alba

Kinetics study of KPC-3, a plasmid-encoded class a carbapenem-hydrolyzing beta-lactamase

ABSTRACT The kinetic activity of KPC-3, a plasmid-encoded class A carbapenemase, was studied. It hydrolyzed penicillins, cephalosporins, carbapenems, and even sulbactam. The best substrate was cephalothin (kcat/Km = 3.48 μM−1 s−1). The efficiency of the enzyme was similar for imipenem and meropenem (kcat/Km, 1.4 and 1.94 μM−1 s−1, respectively).

Extended-Spectrum β-Lactamase-Producing Shiga Toxin Gene (stx1)-Positive Escherichia coli O26:H11: a New Concern

ABSTRACT Escherichia coli strain TUM2139 was isolated from a stool sample from a 9-year-old girl on 16 June 2004. This strain was categorized as Shiga toxin-producing Escherichia coli (STEC) because the Shiga-like toxin gene stx1 was detected by immunochromatography and PCR assay. The strain was highly resistant to cefotaxime (256 μg/ml) and was also resistant to cefepime, cefpodoxime, ceftriaxone, and aztreonam. In the presence of 4 μg of clavulanic acid per ml, the MIC of cefotaxime decreased to ≤0.12 μg/ml, indicating that this strain was an extended-spectrum β-lactamase (ESBL) producer. Cefotaxime resistance was transferred to E. coli C600 by conjugation at a frequency of 3.0 × 10−6. A PCR assay was performed with primer sets specific for TEM-type and SHV-type ESBLs and for the CTX-M-2 (Toho-1), CTX-M-3, and CTX-M-9 groups of ESBLs. A specific signal was observed with the primer set specific for the CTX-M-9 group of β-lactamases. This β-lactamase was confirmed to be the ESBL CTX-M-18 by DNA sequencing. This is the first report of an ESBL-producing STEC isolate.

Clonal Diversity of Metallo-β-Lactamase-Possessing Pseudomonas aeruginosa in Geographically Diverse Regions of Japan

ABSTRACT The aim of this study was to determine the distribution of metallo-β-lactamase-producing Pseudomonas aeruginosa in Japan and to investigate the molecular characteristics of resistance gene cassettes including the gene encoding this enzyme. A total of 594 nonduplicate strains of P. aeruginosa isolated from 60 hospitals throughout Japan in 2002 were evaluated. This study indicated that although the prevalence of imipenem-resistant P. aeruginosa has not increased compared to that found in previous studies, clonal distribution of the same strain across Japan is evident.

Role of a Mutation at Position 167 of CTX-M-19 in Ceftazidime Hydrolysis

ABSTRACT CTX-M-19 is a recently identified ceftazidime-hydrolyzing extended-spectrum β-lactamase, which differs from the majority of CTX-M-type β-lactamases that preferentially hydrolyze cefotaxime but not ceftazidime. To elucidate the mechanism of ceftazidime hydrolysis by CTX-M-19, the β-lactam MICs of a CTX-M-19 producer, and the kinetic parameters of the enzyme were confirmed. We reconfirmed here that CTX-M-19 is also stable at a high enzyme concentration in the presence of bovine serum albumin (20 μg/ml). Under this condition, we obtained more accurate kinetic parameters and determined that cefotaxime (kcat/Km, 1.47 × 106 s−1 M−1), cefoxitin (kcat/Km, 62.2 s−1 M−1), and aztreonam (kcat/Km, 1.34 × 103 s−1 M−1) are good substrates and that imipenem (k+2/K, 1.20 × 102 s−1 M−1) is a poor substrate. However, CTX-M-18 and CTX-M-19 exhibited too high a Km value (2.7 to 5.6 mM) against ceftazidime to obtain their catalytic activity (kcat). Comparison of the MICs with the catalytic efficiency (kcat/Km) of these enzymes showed that some β-lactams, including cefotaxime, ceftazidime, and aztreonam showed a similar correlation. Using the previously reported crystal structure of the Toho-1 β-lactamase, which belongs to the CTX-M-type β-lactamase group, we have suggested characteristic interactions between the enzymes and the β-lactams ceftazidime, cefotaxime, and aztreonam by molecular modeling. Aminothiazole-bearing β-lactams require a displacement of the aminothiazole moiety due to a severe steric interaction with the hydroxyl group of Ser167 in CTX-M-19, and the displacement affects the interaction between Ser130 and the acidic group such as carboxylate and sulfonate of β-lactams.

Kinetic properties of four plasmid-mediated AmpC beta-lactamases.

ABSTRACT The heterologous production in Escherichia coli, the purification, and the kinetic characterization of four plasmid-encoded class C β-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except for their instability, these enzymes are very similar to the known chromosomally encoded AmpC β-lactamases. Their kinetic parameters did not show major differences from those obtained for the corresponding chromosomal enzymes. However, the Km values of CMY-2 for cefuroxime, cefotaxime, and oxacillin were significantly decreased compared to those of the chromosomal AmpC enzymes. Finally, the susceptibility patterns of different E. coli hosts producing a plasmid- or a chromosome-encoded class C enzyme toward β-lactam antibiotics are mainly due to the overproduction of the β-lactamase in the periplasmic space of the bacteria rather than to a specific catalytic profile of the plasmid-encoded β-lactamases.

Novel SHV-Derived Extended-Spectrum β-Lactamase, SHV-57, That Confers Resistance to Ceftazidime but Not Cefazolin

ABSTRACT A new SHV-derived extended-spectrum β-lactamase, SHV-57, that confers high-level resistance to ceftazidime but not cefotaxime or cefazolin was identified from a national surveillance study conducted in Taiwan in 1998. An Escherichia coli isolate resistant to ampicillin, cephalothin, and ceftazidime but sensitive to cefoxitin, ceftriaxone, cefotaxime, imipenem, and a narrow-spectrum cephem (cefazolin) was isolated from the urine of a patient treated with β-lactam antibiotics. Resistance to β-lactams was conjugatively transferred with a plasmid of about 50 kbp. The pI of this enzyme was 8.3. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 861 bases (GenBank accession number AY223863 ). Kinetic parameters showed that SHV-57 had a poor affinity to cefazolin. The Km value toward cefazolin (5.57 × 103 μM) was extremely high in comparison to those toward ceftazidime (30.9 μM) and penicillin G (67 μM), indicating its low affinity to cefazolin. Although the Km value of the β-lactamase inhibitor was too high for the study of catalytic activity (kcat), indicating the low kcat of SHV-57, the SHV-57 carrier was highly susceptible to a β-lactam-β-lactamase inhibitor combination. Comparison of the three-dimensional molecular model of SHV-57 with that of the SHV-1 β-lactamase suggests that the substitution of arginine for leucine-169 in the Ω loop is important for the substrate specificity.

A detailed kinetic study of Mox-1, a plasmid-encoded class C β-lactamase

Abstract Surveys of β-lactamases in different parts of the world show an important increase in class C β-lactamases, thus the study of these enzymes is becoming an important issue. We created an overproduction system for Mox-1, a plasmid class C β-lactamase, by cloning the gene encoding this enzyme, and placing it under the control of a T7 promoter, using vector pET 28a. The enzyme, purified by ion exchange chromatography, was used to obtain the molecular mass (38 246), the N-terminal sequence (GEASPVDPLRPVV), and p I (8.9), and to perform a detailed kinetic study. Cephalotin was used as reporter substrate in the case of poor substrates. The kinetic study showed that benzylpenicillin, cephalotin, cefcapene and moxalactam were good substrates for Mox-1 ( k cat / K m values >2.5 ×10 6 M −1 s −1 ). On the other hand, ceftazidime and cefepime were poor substrates for this enzyme ( K m values >200 μM). Clavulanic acid had no inhibitory effect on Mox-1 ( K m =30.2 mM), however aztreonam behaved as an inhibitor of Mox-1 ( K i =2.85 μM).