Structure and Molecular Recognition Mechanism of IMP-13 Metallo-?-Lactamase.
ABSTRACT: Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-?-lactamases (MBLs) target the most widely used antibiotic class, the ?-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium Pseudomonas aeruginosa found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-?-lactamase inhibitors, essential in the fight against antibiotic resistance.
Project description:Background:Carbapenem resistance in Acinetobacter baumannii has become a major concern for treating physicians. The aim of this study was to investigate the prevalence of metallo ?-lactamase (MBL) genes (bla VIM , and bla IMP) among isolated multidrug-resistant A. baumannii . Methods:Fifty non-repetitive carbapenem-resistant A. baumannii isolates were collected. Antibiotic susceptibility was performed by disk diffusion method. MICs were determined by E test method. The resistant strains were tested for the production of carbapenemases by the Modified Hodge Test (MHT) followed by EDTA-disk synergy test was performed for metallo-?-lactamases (MBL) phenotypic detection. Detection of bla VIM , and bla IMP was performed by PCR followed by sequencing. Results:All isolates had a multidrug resistant profile, and were all resistant to all antibiotics including the carbapenems but remained susceptible to colistin. Among these isolates, Carbapenemase production was confirmed by the Modified Hodge test for 42 (84%) isolates. Phenotypic method showed the production of MBL in 15 (30%) isolates. PCR techniques revealed that out of 50 isolates, 13 (26%) were positive for bla VIM and all were negative for bla IMP . Conclusion:Our study concludes that the high prevalence of carbapenem resistant Acinetobacter species with MBL production is one of the main concerns in our country and this situation needs strict infection control measures.
Project description:The emergence of bacteria that co-express serine- and metallo- carbapenemases is a threat to the efficacy of the available ?-lactam antibiotic armamentarium. The 4-amino-1,2,4-triazole-3-thione scaffold has been selected as the starting chemical moiety in the design of a small library of ?-Lactamase inhibitors (BLIs) with extended activity profiles. The synthesised compounds have been validated in vitro against class A serine ?-Lactamase (SBLs) KPC-2 and class B1 metallo ?-Lactamases (MBLs) VIM-1 and IMP-1. Of the synthesised derivatives, four compounds showed cross-class micromolar inhibition potency and therefore underwent in silico analyses to elucidate their binding mode within the catalytic pockets of serine- and metallo-BLs. Moreover, several members of the synthesised library have been evaluated, in combination with meropenem (MEM), against clinical strains that overexpress BLs for their ability to synergise carbapenems.
Project description:We recently detected a novel variant of an IMP-type metallo-?-lactamase gene (bla IMP-68) from meropenem-resistant but imipenem-susceptible Klebsiella pneumoniae TA6363 isolated in Tokyo, Japan. bla IMP-68 encodes a Ser262Gly point mutant of IMP-11, and transformation experiments showed that bla IMP-68 increased the MIC of carbapenems in recipient strains, whereas the MIC of imipenem was not greatly increased relative to that of other carbapenems, including meropenem. Kinetics experiments showed that IMP-68 imipenem-hydrolyzing activity was lower than that for other carbapenems, suggesting that the antimicrobial susceptibility profile of TA6363 originated from IMP-68 substrate specificity. Whole-genome sequencing showed that bla IMP-68 is harbored by the class 1 integron located on the IncL/M plasmid pTMTA63632 (88,953?bp), which was transferable via conjugation. The presence of plasmid-borne bla IMP-68 is notable, because it conferred antimicrobial resistance to carbapenems, except for imipenem, on Enterobacteriaceae and will likely affect treatment plans using antibacterial agents in clinical settings.IMPORTANCE IMP-type metallo-?-lactamases comprise one group of the "Big 5" carbapenemases. Here, a novel bla IMP-68 gene encoding IMP-68 (harboring a Ser262Gly point mutant of IMP-11) was discovered from meropenem-resistant but imipenem-susceptible Klebsiella pneumoniae TA6363. The Ser262Gly substitution was previously identified as important for substrate specificity according to a study of other IMP variants, including IMP-6. We confirmed that IMP-68 exhibited weaker imipenem-hydrolyzing activity than that for other carbapenems, demonstrating that the antimicrobial susceptibility profile of TA6363 originated from IMP-68 substrate specificity, with this likely to affect treatment strategies using antibacterial agents in clinical settings. Notably, the carbapenem resistance conferred by IMP-68 was undetectable based on the MIC of imipenem as a carbapenem representative, which demonstrates a comparable antimicrobial susceptibility profile to IMP-6-producing Enterobacteriaceae that previously spread in Japan due to lack of awareness of its existence.
Project description:The trends and types of carbapenemase-producing Gram-negative bacilli were analyzed from clinical specimens collected between 2005 and 2012 at a Korean teaching hospital. The proportions of carbapenem-resistant Acinetobacter spp. increased markedly to 66%. Metallo-?-lactamase producers significantly decreased and the majority shifted from the bla(VIM-2) type to the bla(IMP-1) type.
Project description:Metallo-?-lactamases (MBLs) are transmissible carbapenemases of increasing prevalence in Gram-negative bacteria among health care facilities worldwide. Control of the further spread of these carbapenem-resistant bacteria relies on clinical microbiological laboratories correctly identifying and classifying the MBLs. In this study, we evaluated a simple and rapid method for detecting IMP, the most prevalent MBL in Japan. We used an immunochromatography (IC) assay for 181 carbapenem-nonsusceptible (CNS) (nonsusceptible to imipenem or meropenem) strains comprising 74 IMP-producing and 33 non-IMP-producing strains of non-glucose-fermenting Gram-negative rods (NFGNR), as well as 64 IMP-producing and 10 non-IMP-producing Enterobacteriaceae strains. The IC assay results were compared to those from the double-disk synergy test (DDST), the MBL Etest, and the modified Hodge test (MHT) (only for Enterobacteriaceae). The IMP type was confirmed by specific PCR and direct sequencing. The IC assay detected all of the IMP-type MBLs, including IMP-1, -2, -6, -7, -10, -11, -19, -20, and -22 and IMP-40, -41, and -42 (new types), with 100% specificity and sensitivity against all strains tested. Although the sensitivity and specificity values for the DDST and MHT were equivalent to those for the IC assay, the MBL Etest was positive for only 87% of NFGNR and 31% of Enterobacteriaceae due to the low MIC of imipenem, causing an indeterminate evaluation. These results indicated that the IC assay might be a useful alternative to PCR for IMP MBL detection screening.
Project description:In the course of surveying for the carbapenem-hydrolyzing metallo-beta-lactamase gene bla(IMP) in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo-beta-lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the gene bla(IMP-3) was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of bla(IMP-3) was identical to that of bla(IMP-1), except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid bla genes were constructed from bla(IMP-3) and bla(IMP-1) by recombinant DNA techniques, and beta-lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of the bla(IMP-3) gene. This indicated that extension of the substrate profile in the metallo-beta-lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene bla(IMP-3) is an ancestor of bla(IMP-1).
Project description:The already considerable global public health threat of multi-drug resistant Gram-negative bacteria has become even more of a concern following the emergence of New-Delhi metallo-?-lactamase (NDM-1) producing strains of Klebsiella pneumoniae and other Gram-negative bacteria. As an alternative approach to the traditional development of new bactericidal entities, we have identified a 2-aminoimidazole derived small molecule that acts as an antibiotic adjuvant and is able to suppress resistance of a NDM-1 producing strain of K. pneumoniae to imipenem and meropenem, in addition to suppressing resistance of other ?-lactam non-susceptible K. pneumoniae strains. The small molecule is able to lower carbapenem minimum inhibitory concentrations by up to 16-fold while exhibiting little bactericidal activity itself.
Project description:In Gram-negative bacteria, resistance to ?-lactam antibacterials is largely due to ?-lactamases and is a growing public health threat. One of the most concerning ?-lactamases to evolve in bacteria are the Class B enzymes, the metallo-?-lactamases (MBLs). To date, penams and cephems resistant to hydrolysis by MBLs have not yet been found. As a result of this broad substrate specificity, a better understanding of the role of catalytically important amino acids in MBLs is necessary to design novel ?-lactams and inhibitors. Two MBLs, the wild type IMP-1 with serine at position 262, and an engineered variant with valine at the same position (IMP-1-S262V), were previously found to exhibit very different substrate spectra. These findings compelled us to investigate the impact of a threonine at position 262 (IMP-1-S262T) on the substrate spectrum. Here, we explore MBL sequence-structure-activity relationships by predicting and experimentally validating the effect of the S262T substitution in IMP-1. Using site-directed mutagenesis, threonine was introduced at position 262, and the IMP-1-S262T enzyme, as well as the other two enzymes IMP-1 and IMP-1-S262V, were purified and kinetic constants were determined against a range of ?-lactam antibacterials. Catalytic efficiencies (kcat /KM ) obtained with IMP-1-S262T and minimum inhibitory concentrations (MICs) observed with bacterial cells expressing the protein were intermediate or comparable to the corresponding values with IMP-1 and IMP-1-S262V, validating the role of this residue in catalysis. Our results reveal the important role of IMP residue 262 in ?-lactam turnover and support this approach to predict activities of certain novel MBL variants.
Project description:In an effort to test whether a transition state analog is an inhibitor of the metallo-?-lactamases, a phospholactam analog of carbapenem has been synthesized and characterized. The phospholactam 1 proved to be a weak, time-dependent inhibitor of IMP-1 (70%), CcrA (70%), L1 (70%), NDM-1 (53%), and Bla2 (94%) at an inhibitor concentration of 100?M. The phospholactam 1 activated ImiS and BcII at the same concentration. Docking studies were used to explain binding and to offer suggestions for modifications to the phospholactam scaffold to improve binding affinities.
Project description:Metallo-?-lactamase-producing Pseudomonas aeruginosa (MPPA) is an important nosocomial pathogen that shows resistance to all ?-lactam antibiotics except monobactams. There are various types of metallo-?-lactamases (MBLs) in carbapenem-resistant P. aeruginosa including Imipenemase (IMP), Verona integron-encoded metallo-?-lactamase (VIM), Sao Paulo metallo-?-lactamase (SPM), Germany imipenemase (GIM), New Delhi metallo-?-lactamase (NDM), Florence imipenemase (FIM). Each MBL gene is located on specific genetic elements including integrons, transposons, plasmids, or on the chromosome, in which they carry genes encoding determinants of resistance to carbapenems and other antibiotics, conferring multidrug resistance to P. aeruginosa. In addition, these genetic elements are transferable to other Gram-negative species, increasing the antimicrobial resistance rate and complicating the treatment of infected patients. Therefore, it is essential to understand the epidemiology, resistance mechanism, and molecular characteristics of MPPA for infection control and prevention of a possible global health crisis. Here, we highlight the characteristics of MPPA.