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Analysis of BlaEC family class C beta-lactamase.

ABSTRACT: Recent years have witnessed an increased prevalence of intrinsic and acquired beta-lactamase-producing bacteria, severely limiting human and veterinary medicine therapeutic options. The present study aimed to design specific oligonucleotides for rapid PCR detection of the cephalosporinase-encoding gene blaEC (BlaEC family class C beta-lactamase). A total of three primers were designed to detect 2281 variants of the blaEC gene and two sets of primer pairs were also tested against DNA from 11 strains. The study indicates that the proposed primers should be able to detect 100% of all described blaEC genes in different bacterial strains and monitor their spread. After comparing the amino acid sequences, a phylogenetic tree was created based on the presence of conserved amino acids and homologous motifs. More than 24 760 mutations in BlaEC enzymes have been identified. The mutations involving 371 amino acid positions and these hotspots can change the structure and activity of the monitored enzymes. We predicted several BlaEC enzymes with a broadened substrate activity against higher-generation cephalosporins.

SUBMITTER: Schmidt J

PROVIDER: S-EPMC10563145 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Analysis of BlaEC family class C beta-lactamase.

Schmidt Jiri J Zdarska Veronika V Kolar Milan M Mlynarcik Patrik P

FEMS microbiology letters 20230101

Recent years have witnessed an increased prevalence of intrinsic and acquired beta-lactamase-producing bacteria, severely limiting human and veterinary medicine therapeutic options. The present study aimed to design specific oligonucleotides for rapid PCR detection of the cephalosporinase-encoding gene blaEC (BlaEC family class C beta-lactamase). A total of three primers were designed to detect 2281 variants of the blaEC gene and two sets of primer pairs were also tested against DNA from 11 stra ...[more]

PMID: 37757475

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Project description:As resistance determinants, KPC beta-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type beta-lactamases are often identified as resistant to standard beta-lactam-beta-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to beta-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (kcat/kinact ratios, where kinact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp2-hybridized C3 carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: Km of penem 1, 0.06+/-0.01 microM, and Km of penem 2, 0.006+/-0.001 microM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (kcat/kinact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (kinact/Km=6.5+/-0.6 microM(-1) s(-1)). We conclude that KPC-2 is unique among class A beta-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type beta-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current beta-lactamase inhibitors.

Dataset Information

Analysis of BlaEC family class C beta-lactamase.

Publications

Analysis of BlaEC family class C beta-lactamase.

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