-
The Indian Journal of Medical Research Nov 2010
Topics: Carbapenems; Drug Resistance, Bacterial; Enterobacteriaceae; Humans; India; beta-Lactamases
PubMed: 21149994
DOI: No ID Found -
Specificity and cooperativity at β-lactamase position 104 in TEM-1/BLIP and SHV-1/BLIP interactions.Proteins Apr 2011Establishing a quantitative understanding of the determinants of affinity in protein-protein interactions remains challenging. For example, TEM-1/β-lactamase inhibitor...
Establishing a quantitative understanding of the determinants of affinity in protein-protein interactions remains challenging. For example, TEM-1/β-lactamase inhibitor protein (BLIP) and SHV-1/BLIP are homologous β-lactamase/β-lactamase inhibitor protein complexes with disparate K(d) values (3 nM and 2 μM, respectively), and a single substitution, D104E in SHV-1, results in a 1000-fold enhancement in binding affinity. In TEM-1, E104 participates in a salt bridge with BLIP K74, whereas the corresponding SHV-1 D104 does not in the wild type SHV-1/BLIP co-structure. Here, we present a 1.6 Å crystal structure of the SHV-1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV-1 D104 and BLIP K74 and a 400-fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network.
Topics: Bacterial Proteins; Binding Sites; Crystallography; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Protein Stability; Thermodynamics; beta-Lactamases
PubMed: 21294157
DOI: 10.1002/prot.22961 -
Biochemical Pharmacology Dec 2007One strategy employed by bacterial strains to resist beta-lactam antibiotics is the expression of metallo-beta-lactamases requiring Zn(2+) for activity. In the last few... (Review)
Review
One strategy employed by bacterial strains to resist beta-lactam antibiotics is the expression of metallo-beta-lactamases requiring Zn(2+) for activity. In the last few years, many new zinc beta-lactamases have been described and several pathogens are now known to synthesize members of this class. Metallo-beta-lactamases are especially worrisome due to: (1) their broad activity profiles that encompass most beta-lactam antibiotics, including the carbapenems; (2) potential for horizontal transference; and (3) the absence of clinically useful inhibitors. On the basis of the known sequences, three different lineages, identified as subclasses B1, B2, and B3 have been characterized. The three-dimensional structure of at least one metallo-beta-lactamase of each subclass has been solved. These very similar 3D structures are characterized by the presence of an alphabetabetaalpha-fold. In addition to metallo-beta-lactamases which cleave the amide bond of the beta-lactam ring, the metallo-beta-lactamase superfamily includes enzymes which hydrolyze thiol-ester, phosphodiester and sulfuric ester bonds as well as oxydoreductases. Most of the 6000 members of this superfamily share five conserved motifs, the most characteristic being the His116-X-His118-X-Asp120-His121 signature. They all exhibit an alphabetabetaalpha-fold, similar to that found in the structure of zinc beta-lactamases. Many members of this superfamily are involved in mRNA maturation and DNA reparation. This fact suggests the hypothesis that metallo-beta-lactamases may be the result of divergent evolution starting from an ancestral protein which did not have a beta-lactamase activity.
Topics: Anti-Bacterial Agents; Catalysis; Enzyme Inhibitors; Microbial Sensitivity Tests; Models, Molecular; Phylogeny; Protein Conformation; Zinc; beta-Lactamases
PubMed: 17597585
DOI: 10.1016/j.bcp.2007.05.021 -
Cellular and Molecular Life Sciences :... Apr 1998beta-Lactamase production is responsible for the appearance of a large number of pathogenic bacterial strains exhibiting a high degree of resistance to beta-lactam... (Review)
Review
beta-Lactamase production is responsible for the appearance of a large number of pathogenic bacterial strains exhibiting a high degree of resistance to beta-lactam antibiotics. A large number of enzymes have been described with very diverse primary structures and catalytic profiles. Nevertheless, all known three-dimensional structures of active-site serine beta-lactamases exhibit a high degree of similarity with apparently equivalent chemical functionalities in the same strategic positions. These groups might not, however, play identical roles in the various classes of enzymes. Structural data have also been recently obtained for the zinc metallo-beta-lactamases, but the detailed catalytic mechanisms might also differ widely, depending on the enzyme studied. Similarly, the induction of the synthesis of beta-lactamases is now better understood, but many questions remain to be answered.
Topics: Gene Expression Regulation, Bacterial; Gram-Negative Bacteria; Gram-Positive Bacteria; Models, Molecular; Serine; Structure-Activity Relationship; Zinc; beta-Lactamases
PubMed: 9614970
DOI: 10.1007/s000180050161 -
International Journal of Infectious... May 2007Plasmid-mediated class C beta-lactamases are reported from Enterobacteriaceae with increasing frequency. They likely originate from chromosomal AmpC of certain... (Review)
Review
Plasmid-mediated class C beta-lactamases are reported from Enterobacteriaceae with increasing frequency. They likely originate from chromosomal AmpC of certain Gram-negative bacterial species and subsequently are mobilized onto transmissible plasmids. There are reports of unfavorable clinical outcomes in patients infected with these organisms and treated with broad-spectrum cephalosporins. However, unlike class A extended-spectrum beta-lactamases (ESBLs), no screening and confirmatory tests have been uniformly established for strains that produce class C beta-lactamases. Reduced susceptibility to cefoxitin is a sensitive but not specific indicator of class C beta-lactamase production. Simple confirmatory tests including tests using boronic acid compounds as specific class C beta-lactamase inhibitors have recently been developed. Their utilization will enable clinical microbiology laboratories to report those strains producing plasmid-mediated class C beta-lactamases as being resistant to all broad-spectrum cephalosporins, thus allowing physicians to prescribe appropriate antimicrobial therapy.
Topics: Anti-Bacterial Agents; Cephalosporins; Enterobacteriaceae; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Microbial Sensitivity Tests; Plasmids; Polymerase Chain Reaction; beta-Lactamase Inhibitors; beta-Lactamases
PubMed: 17339123
DOI: 10.1016/j.ijid.2006.07.008 -
Journal of Biotechnology Aug 2018β-lactams are one of the most common antimicrobials used to treat bacterial infections. However, bacterial resistance has compromised their efficacy, mainly due to the...
β-lactams are one of the most common antimicrobials used to treat bacterial infections. However, bacterial resistance has compromised their efficacy, mainly due to the β-lactamase enzyme production. To overcome this resistance, β-lactamase inhibitors can be used in association with these antimicrobials. Herein, we analyzed the structural characteristics of β-lactamases and their interactions with classical inhibitors, such as clavulanic acid (CA), sulbactam (SB) and tazobactam (TZ) to gain insights into resistance. The homology models of five class A β-lactamases, namely CARB-3, IMI-1, SFO-1, SHV-5 and TEM-10, were constructed and validated and revealed an overall 3D structural conservation, but with significant differences in the electrostatic potential maps, especially at important regions in the catalytic site. Molecular dockings of CA, SB and TZ with these enzymes revealed a covalent bond with the S70 in all complexes, except Carb-3 which is in agreement with experimental data reported so far. This is likely related to the less voluminous active site of Carb-3 model. Although few specific contacts were observed in the β-lactamase-inhibitor complexes, all compounds interacted with the residues in positions 73, 130, 132, 236 and 237. Therefore, this study provides new perspectives for the design of innovative compounds with broad-spectrum inhibitory profiles against β-lactamases.
Topics: Amino Acid Sequence; Bacterial Proteins; Molecular Docking Simulation; Protein Binding; Sequence Alignment; beta-Lactam Resistance; beta-Lactamase Inhibitors; beta-Lactamases
PubMed: 29753682
DOI: 10.1016/j.jbiotec.2018.05.005 -
Antimicrobial Agents and Chemotherapy Sep 1997Amino acid changes that influence activity and resistance to beta-lactams and beta-lactamase inhibitors were explored by constructing the Gly238Ser and...
Amino acid changes that influence activity and resistance to beta-lactams and beta-lactamase inhibitors were explored by constructing the Gly238Ser and Met69Ile-Gly238Ser mutants of the OHIO-1 beta-lactamase, a class A enzyme of the SHV family. The Km values of cefotaxime and ceftazidime for OHIO-1 and Met69Ile beta-lactamases were > or = 500 microM. The Km of cefotaxime for the Gly238Ser beta-lactamase was 26 microM, and that of ceftazidime was 105 microM. The Km of cefotaxime for the Met69Ile-Gly238Ser beta-lactamase was 292 microM, and that of ceftazidime was 392 microM. For the beta-lactamase inhibitors clavulanate and sulbactam, the apparent Ki values for the Met69Ile-Gly238Ser enzyme were 0.03 and 0.15 microM, respectively. Relative Vmax values indicate that the Met69Ile-Gly238Ser mutant of the OHIO-1 beta-lactamase possesses cephalosporinase activity similar to that of the Gly238Ser mutant but diminished penicillinase activity. In an Escherichia coli DH5alpha strain that possesses a Met69Ile beta-lactamase of the OHIO-1 family, the added Gly238Ser mutation resulted in a phenotype with qualities that confer resistance to expanded-spectrum cephalosporins and, to a lesser extent, beta-lactamase inhibitors.
Topics: Cephalosporin Resistance; Enzyme Inhibitors; Glycine; Isoleucine; Kinetics; Methionine; Mutagenesis, Site-Directed; Mutation; Phenotype; Serine; beta-Lactam Resistance; beta-Lactamases
PubMed: 9303389
DOI: 10.1128/AAC.41.9.1940 -
Clinical Microbiology Reviews Jan 2016For medical biologists, sequencing has become a commonplace technique to support diagnosis. Rapid changes in this field have led to the generation of large amounts of... (Review)
Review
For medical biologists, sequencing has become a commonplace technique to support diagnosis. Rapid changes in this field have led to the generation of large amounts of data, which are not always correctly listed in databases. This is particularly true for data concerning class A β-lactamases, a group of key antibiotic resistance enzymes produced by bacteria. Many genomes have been reported to contain putative β-lactamase genes, which can be compared with representative types. We analyzed several hundred amino acid sequences of class A β-lactamase enzymes for phylogenic relationships, the presence of specific residues, and cluster patterns. A clear distinction was first made between dd-peptidases and class A enzymes based on a small number of residues (S70, K73, P107, 130SDN132, G144, E166, 234K/R, 235T/S, and 236G [Ambler numbering]). Other residues clearly separated two main branches, which we named subclasses A1 and A2. Various clusters were identified on the major branch (subclass A1) on the basis of signature residues associated with catalytic properties (e.g., limited-spectrum β-lactamases, extended-spectrum β-lactamases, and carbapenemases). For subclass A2 enzymes (e.g., CfxA, CIA-1, CME-1, PER-1, and VEB-1), 43 conserved residues were characterized, and several significant insertions were detected. This diversity in the amino acid sequences of β-lactamases must be taken into account to ensure that new enzymes are accurately identified. However, with the exception of PER types, this diversity is poorly represented in existing X-ray crystallographic data.
Topics: Bacteria; Crystallography, X-Ray; Genetic Variation; Genotype; Phylogeny; Protein Conformation; Structure-Activity Relationship; beta-Lactamases
PubMed: 26511485
DOI: 10.1128/CMR.00019-15 -
Journal of Global Antimicrobial... Dec 2020Many multidrug-resistant Gram-negative bacilli (MDR-GNB) harbour multiple β-lactamases. The aim of this study was to assess the impact of multiple β-lactamase carriage...
Does the presence of multiple β-lactamases in Gram-negative bacilli impact the results of antimicrobial susceptibility tests and extended-spectrum β-lactamase and carbapenemase confirmation methods?
OBJECTIVES
Many multidrug-resistant Gram-negative bacilli (MDR-GNB) harbour multiple β-lactamases. The aim of this study was to assess the impact of multiple β-lactamase carriage on the accuracy of susceptibility tests and extended-spectrum β-lactamase (ESBL) and carbapenemase confirmation methods.
METHODS
A total of 50 MDR-GNB, of which 29 carried multiple β-lactamases, underwent broth microdilution (BMD) and disk diffusion (DD) testing as well as confirmation tests for ESBLs and carbapenemases. Whole-genome sequencing (WGS) was used for β-lactamase gene identification.
RESULTS
Categorical agreement of BMD and DD testing results ranged from 86.5 to 97.7% for 10 β-lactam agents. BMD and DD algorithms for ESBL detection were highly variable; 6 of 8 positive strains carried an ESBL plus a carbapenemase or an AmpC enzyme, which may confound antimicrobial selection. The sensitivity and specificity of the modified carbapenem inactivation method (mCIM) were both 100%, whilst mCIM and EDTA-modified carbapenem inactivation method (eCIM) when used together to differentiate serine from metallo-β-lactamase carriage were both 96%. Xpert® Carba-R results (in vitro diagnostic test) were consistent with WGS results. Predicting phenotypic carbapenem resistance from WGS data overall showed 100% specificity but only 66.7% sensitivity for Enterobacterales isolates that were non-susceptible to imipenem and meropenem.
CONCLUSIONS
Multiple β-lactamases in MDR-GNB does not impact DD results, the utility of mCIM/eCIM tests, or Xpert Carba-R results. However, ESBL algorithms produced inconsistent results and predicting carbapenem resistance from WGS data was problematic in such strains.
Topics: Bacterial Proteins; Diagnostic Tests, Routine; Gram-Negative Bacteria; beta-Lactamases
PubMed: 32889141
DOI: 10.1016/j.jgar.2020.08.011 -
Central European Journal of Public... Jun 2022The beta-lactamases with extended spectrum of activity (ESBL) are medically one of the most important group of enzymes. Another group of beta-lactamases representing of...
OBJECTIVES
The beta-lactamases with extended spectrum of activity (ESBL) are medically one of the most important group of enzymes. Another group of beta-lactamases representing of Enterobacteriaceae is group of the AmpC-type cephalosporinases. The presented study provides identification and determination of the spectrum of resistance against different and clinically used antimicrobial drugs in the clinical isolates of Escherichia coli.
METHODS
These isolates had origin in different departments of the L. Pasteur University Hospital in Košice. The goal was the detection of beta-lactamase production with extended-spectrum effect and testing of AmpC-type cephalosporinases by several phenotypic tests in clinical isolates. MALDI-TOF MS analysis was performed on a Microflex MALDI Biotyper. Samples were positively tested for ESBL with the use of the disc diffusion method. PCR were performed with a series of primers designed for the detection of Ambler class A, B and C beta-lactamase genes.
RESULTS
For all 485 isolates, we determined the production of ESBL, which we detected in 166 E. coli isolates, which represents a 34.2% prevalence of ESBL production. It is clear from the results that the prevalence of ESBL-producing E. coli out of the total number of E. coli investigated reached 34.2%. In the monitored period, we confirmed at least one resistance gene from 485 E. coli in 188 positive isolates.
CONCLUSIONS
We describe a complex ESBL epidemiology. The study revealed a high rate of ESBL-producing E. coli isolates; bla and bla enzymes dominated in ESBL-positive E. coli isolates in the L. Pasteur University Hospital in Košice.
Topics: Anti-Bacterial Agents; Bacteria; Drug Resistance, Microbial; Escherichia coli; Escherichia coli Infections; Humans; beta-Lactamases
PubMed: 35841230
DOI: 10.21101/cejph.a7384