scholarly journals Relebactam Is a Potent Inhibitor of the KPC-2 β-Lactamase and Restores Imipenem Susceptibility in KPC-Producing Enterobacteriaceae

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Melissa D. Barnes ◽  
Jim Alsop ◽  
Magdalena A. Taracila ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Rate Constant ◽  
Active Site ◽  
Klebsiella Oxytoca ◽  
Enterobacter Aerogenes ◽  
Clinical Isolates ◽  
Piperidine Ring ◽  
Water Molecules ◽  
Content Type ◽  
Site Water

ABSTRACT The imipenem-relebactam combination is in development as a potential treatment regimen for infections caused by Enterobacteriaceae possessing complex β-lactamase backgrounds. Relebactam is a β-lactamase inhibitor that possesses the diazabicyclooctane core, as in avibactam; however, the R1 side chain of relebactam also includes a piperidine ring, whereas that of avibactam is a carboxyamide. Here, we investigated the inactivation of the Klebsiella pneumoniae carbapenemase KPC-2, the most widespread class A carbapenemase, by relebactam and performed susceptibility testing with imipenem-relebactam using KPC-producing clinical isolates of Enterobacteriaceae . MIC measurements using agar dilution methods revealed that all 101 clinical isolates of KPC-producing Enterobacteriaceae ( K. pneumoniae , Klebsiella oxytoca , Enterobacter cloacae , Enterobacter aerogenes , Citrobacter freundii , Citrobacter koseri , and Escherichia coli ) were highly susceptible to imipenem-relebactam (MICs ≤ 2 mg/liter). Relebactam inhibited KPC-2 with a second-order onset of acylation rate constant ( k 2 / K ) value of 24,750 M −1 s −1 and demonstrated a slow off-rate constant ( k off ) of 0.0002 s −1 . Biochemical analysis using time-based mass spectrometry to map intermediates revealed that the KPC-2–relebactam acyl-enzyme complex was stable for up to 24 h. Importantly, desulfation of relebactam was not observed using mass spectrometry. Desulfation and subsequent deacylation have been observed during the reaction of KPC-2 with avibactam. Upon molecular dynamics simulations of relebactam in the KPC-2 active site, we found that the positioning of active-site water molecules is less favorable for desulfation in the KPC-2 active site than it is in the KPC-2–avibactam complex. In the acyl complexes, the water molecules are within 2.5 to 3 Å of the avibactam sulfate; however, they are more than 5 to 6 Å from the relebactam sulfate. As a result, we propose that the KPC-2–relebactam acyl complex is more stable than the KPC-2–avibactam complex. The clinical implications of this difference are not currently known.

scholarly journals A Prospective Study of Acinetobacter baumannii Complex Isolates and Colistin Susceptibility Monitoring by Mass Spectrometry of Microbial Membrane Glycolipids

2018 ◽  
Vol 57 (3) ◽  
Author(s):  
Lisa M. Leung ◽  
Christi L. McElheny ◽  
Francesca M. Gardner ◽  
Courtney E. Chandler ◽  
Sarah L. Bowler ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Acinetobacter Baumannii ◽  
Lipid A ◽  
Membrane Lipids ◽  
Clinical Isolates ◽  
Colistin Resistance ◽  
Hospital System ◽  
Content Type ◽  
Acinetobacter Baumannii Complex ◽  
Gram Negative Organisms

ABSTRACT Acinetobacter baumannii is a prevalent nosocomial pathogen with a high incidence of multidrug resistance. Treatment of infections due to this organism with colistin, a last-resort antibiotic of the polymyxin class, can result in the emergence of colistin-resistant strains. Colistin resistance primarily occurs via modifications of the terminal phosphate moieties of lipopolysaccharide-derived lipid A, which reduces overall membrane electronegativity. These modifications are readily identified by mass spectrometry (MS). In this study, we prospectively collected Acinetobacter baumannii complex clinical isolates from a hospital system in Pennsylvania over a 3-year period. All isolates were evaluated for colistin resistance using standard MIC testing by both agar dilution and broth microdilution, as well as genospecies identification and lipid A profiling using MS analyses. Overall, an excellent correlation between colistin susceptibility and resistance, determined by MIC testing, and the presence of a lipid A modification, determined by MS, was observed with a sensitivity of 92.9% and a specificity of 94.0%. Additionally, glycolipid profiling was able to differentiate A. baumannii complex organisms based on their membrane lipids. With the growth of MS use in clinical laboratories, a reliable MS-based glycolipid phenotyping method that identifies colistin resistance in A. baumannii complex clinical isolates, as well as other Gram-negative organisms, represents an alternative or complementary approach to existing diagnostics.

scholarly journals Surveillance of Omadacycline Activity against Clinical Isolates from a Global Collection (North America, Europe, Latin America, Asia-Western Pacific), 2010-2011

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Michael A. Pfaller ◽  
Michael D. Huband ◽  
Paul R. Rhomberg ◽  
Robert K. Flamm
Keyword(s):  
Latin America ◽  
North America ◽  
Klebsiella Oxytoca ◽  
Enterobacter Aerogenes ◽  
Community Acquired Pneumonia ◽  
Asia Pacific ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Viridans Group Streptococci

ABSTRACT Omadacycline is a broad-spectrum aminomethylcycline in late-stage clinical development for the treatment of acute bacterial skin and skin structure infections and community-acquired pneumonia as an oral and an intravenous once-daily formulation. In this study, omadacycline and comparators were tested against 69,246 nonduplicate bacterial isolates collected prospectively during 2010 and 2011 from medical centers in Asia-Pacific (11,397 isolates), Europe (23,490 isolates), Latin America (8,038 isolates), and North America (26,321 isolates). Omadacycline was tested by broth microdilution following Clinical and Laboratory Standards Institute M07-A10 (2015) methods. A total of 99.9% of Staphylococcus aureus isolates were inhibited by ≤2 μg/ml of omadacycline (MIC50/90, 0.12/0.25 μg/ml), including 100.0% of methicillin-susceptible S. aureus isolates and 99.8% of methicillin-resistant S. aureus isolates. Omadacycline potencies were comparable for Streptococcus pneumoniae (MIC50/90, 0.06/0.06 μg/ml), viridans group streptococci (MIC50/90, 0.06/0.12 μg/ml), and beta-hemolytic streptococci (MIC50/90, 0.06/0.12 μg/ml) regardless of species and susceptibility to penicillin. Omadacycline was active against Enterobacteriaceae and was most active against Escherichia coli (MIC50/90, 0.5/2 μg/ml), Enterobacter aerogenes (MIC50/90, 2/4 μg/ml), Klebsiella oxytoca (MIC50/90, 1/4 μg/ml), and Citrobacter spp. (MIC50/90, 1/4 μg/ml). Omadacycline was active against Haemophilus influenzae (MIC50/90, 1/1 μg/ml) regardless of β-lactamase status and against Moraxella catarrhalis (MIC50/90, 0.12/0.25 μg/ml). The potent activity of omadacycline against Gram-positive and Gram-negative bacteria indicates that omadacycline merits further study in serious infections in which multidrug resistance and mixed Gram-positive and Gram-negative infections may be a concern.

scholarly journals Role of the Outer Membrane and Porins in Susceptibility of β-Lactamase-Producing Enterobacteriaceae to Ceftazidime-Avibactam

2015 ◽  
Vol 60 (3) ◽  
pp. 1349-1359 ◽  
Author(s):  
Jean-Marie Pagès ◽  
Sabine Peslier ◽  
Thomas A. Keating ◽  
Jean-Philippe Lavigne ◽  
Wright W. Nichols
Keyword(s):  
Membrane Permeability ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Enterobacter Aerogenes ◽  
Antibacterial Activities ◽  
Clinical Isolates ◽  
Membrane Pore ◽  
Content Type ◽  
Porin Proteins ◽  
Outer Membrane Permeability

This study examined the activity of the novel antimicrobial combination ceftazidime-avibactam againstEnterobacteriaceaeexhibiting different outer membrane permeability profiles, specifically with or without porins and with or without expression of the main efflux pump (AcrAB-TolC). The addition of the outer membrane permeabilizer polymyxin B nonapeptide increased the antibacterial activities of avibactam alone, ceftazidime alone, and ceftazidime-avibactam against the characterized clinical isolates ofEscherichia coli,Enterobacter aerogenes, andKlebsiella pneumoniae. This enhancement of activities was mainly due to increased passive penetration of compounds since inhibition of efflux by the addition of phenylalanine-arginine β-naphthylamide affected the MICs minimally. OmpF (OmpK35) or OmpC (OmpK36) pores were not the major route by which avibactam crossed the outer membranes ofE. coliandK. pneumoniae. In contrast, Omp35 and Omp36 allowed diffusion of avibactam across the outer membrane ofE. aerogenes, although other diffusion channels for avibactam were also present in that species. It was clear that outer membrane permeability and outer membrane pore-forming proteins play a key role in the activity of ceftazidime-avibactam. Nevertheless, the MICs of ceftazidime-avibactam (with 4 mg/liter avibactam) against the ceftazidime-resistant clinical isolates of the three species ofEnterobacteriaceaestudied were ≤8 mg/liter, regardless of outer membrane permeability changes resulting from an absence of defined porin proteins or upregulation of efflux.

scholarly journals Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Melina Ruggiero ◽  
Krisztina M. Papp-Wallace ◽  
Magdalena A. Taracila ◽  
Maria F. Mojica ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Active Site ◽  
Therapeutic Option ◽  
Structural Features ◽  
Atomic Mass ◽  
Stable Complex ◽  
Water Molecules ◽  
Gram Negative Bacteria ◽  
Biophysical Properties ◽  
Content Type ◽  
Class A

ABSTRACT PER β-lactamases are an emerging family of extended-spectrum β-lactamases (ESBL) found in Gram-negative bacteria. PER β-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 β-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k 2/K = 2 × 103 ± 0.1 × 103 M−1 s−1, where k 2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D β-lactamases (i.e., k 2/K range of ≈103 M−1 s−1) and not class A β-lactamases (i.e., k 2/K range, 104 to 105 M−1 s−1). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k 2/K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested different β-lactam–AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing bla PER-2. Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation.

scholarly journals Dissemination of Clinical Isolates of Klebsiella oxytoca Harboring CMY-31, VIM-1, and a New OXY-2-Type Variant in the Community

2011 ◽  
Vol 55 (7) ◽  
pp. 3164-3168 ◽  
Author(s):  
Athanassios Tsakris ◽  
Aggeliki Poulou ◽  
Fani Markou ◽  
Vassiliki Pitiriga ◽  
Evangelia-Theophano Piperaki ◽  
...  
Keyword(s):  
Klebsiella Oxytoca ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Health Care Facilities ◽  
Clinical Isolates ◽  
Nucleotide Sequencing ◽  
Class 1 Integron ◽  
Content Type ◽  
Clonal Type ◽  
Tract Infections

ABSTRACTThe aim of the present study was to investigate the epidemiological link of multidrug-resistantKlebsiella oxytocaisolates causing community-onset infections among patients attending our outpatient department and to investigate the underlying resistance mechanisms. The isolates were tested by agar dilution MICs, phenotypic carbapenemase testing, enterobacterial repetitive intergenic consensus-PCR, and pulsed-field gel electrophoresis (PFGE). PCR assays and nucleotide sequencing were employed for the identification ofblagene types and the mapping of the integron-containing metallo-β-lactamase (MBL) gene. During the study period (January 2005 to April 2007), nine broad-spectrum cephalosporin-resistantK. oxytocaclinical isolates were prospectively collected from separate outpatients with urinary tract infections. In all cases, the patients had been hospitalized or exposed to health care facilities during the preceding year. Molecular typing revealed that all isolates belonged to the sameK. oxytocaclonal type, which contained five PFGE subtypes. A novel chromosomal OXY-2 β-lactamase type variant (OXY-2-9) was detected in all isolates, but no mutations in the promoter region justifyingblaOXYgene overproduction were detected. In addition, all isolates harbored the plasmidic CMY-31 (LAT-4) AmpC cephalosporinase, while three of them harbored VIM-1 MBL in a class 1 integron structure. This is the first study to present the dissemination in the community of multidrug-resistantK. oxytocaisolates causing extrahospital infections.

scholarly journals Frequency and Mechanism of Spontaneous Resistance to Sulbactam Combined with the Novel β-Lactamase Inhibitor ETX2514 in Clinical Isolates of Acinetobacter baumannii

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Sarah M. McLeod ◽  
Adam B. Shapiro ◽  
Samir H. Moussa ◽  
Michele Johnstone ◽  
Robert E. McLaughlin ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Broad Spectrum ◽  
Active Site ◽  
Stringent Response ◽  
Clinical Isolates ◽  
The Novel ◽  
Penicillin Binding Protein ◽  
Content Type ◽  
Sensitive Isolate ◽  
Lactamase Inhibitor

ABSTRACTThe novel diazabicyclooctenone ETX2514 is a potent, broad-spectrum serine β-lactamase inhibitor that restores sulbactam activity against resistantAcinetobacter baumannii. The frequency of spontaneous resistance to sulbactam-ETX2514 in clinical isolates was found to be 7.6 × 10−10to <9.0 × 10−10at 4× MIC and mapped to residues near the active site of penicillin binding protein 3 (PBP3). Purified mutant PBP3 proteins demonstrated reduced affinity for sulbactam. In a sulbactam-sensitive isolate, resistance also mapped to stringent response genes associated with resistance to PBP2 inhibitors, suggesting that in addition to β-lactamase inhibition, ETX2514 may enhance sulbactam activity inA. baumanniivia inhibition of PBP2.

scholarly journals Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 22 ◽  
Author(s):  
Ignacio Boron ◽  
Juan Pablo Bustamante ◽  
Kelly S Davidge ◽  
Sandip Singh ◽  
Lesley AH Bowman ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Water Molecules ◽  
Single Mutation ◽  
Truncated Hemoglobin ◽  
Kinetic Measurements ◽  
Migration Rates ◽  
Ligand Migration ◽  
Site Water ◽  
Dynamics Simulations

Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O2 and •NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify •NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, •NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

scholarly journals Distribution of Extended-Spectrum β-Lactamases, AmpC β-Lactamases, and Carbapenemases among Enterobacteriaceae Isolates Causing Intra-Abdominal Infections in the Asia-Pacific Region: Results of the Study for Monitoring Antimicrobial Resistance Trends (SMART)

2013 ◽  
Vol 57 (7) ◽  
pp. 2981-2988 ◽  
Author(s):  
Wang-Huei Sheng ◽  
Robert E. Badal ◽  
Po-Ren Hsueh
Keyword(s):  
Antimicrobial Resistance ◽  
Klebsiella Oxytoca ◽  
Enterobacter Aerogenes ◽  
Asia Pacific ◽  
Pacific Region ◽  
Asia Pacific Region ◽  
Content Type ◽  
Extended Spectrum ◽  
Abdominal Infections ◽  
Enterobacter Asburiae

ABSTRACTThe increasing trend of β-lactam resistance amongEnterobacteriaceaeis a worldwide threat.Enterobacteriaceaeisolates causing intra-abdominal infections (IAI) from the Study for Monitoring Antimicrobial Resistance Trends (SMART) collected in 2008 and 2009 from the Asia-Pacific region were investigated. Detection of extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases was performed by multiplex PCR. A total of 699Enterobacteriaceaeisolates with positive genotypic results, includedEscherichia coli(n= 443),Klebsiella pneumoniae(n= 187),Enterobacter cloacae(n= 45),Klebsiella oxytoca(n= 9),Citrobacter freundii(n= 5),Proteus mirabilis(n= 3),Enterobacter aerogenes(n= 2),Morganella morganii(n= 2), and one each ofEnterobacter asburiae,Proteus vulgaris, andProvidencia rettgeriwere analyzed. Nearly 20% of these β-lactamase-producingEnterobacteriaceaeisolates were from community-associated IAI. CTX-M (588 isolates, including 428 [72.8%] with CTX-M-15) was the most common ESBL, followed by SHV (n= 59) and TEM (n= 4). CMY (n= 110, including 102 [92.7%] with CMY-2) was the most common AmpC β-lactamase, followed by DHA (n= 46) and ACT/MIR (n= 40). NDM (n= 65, including 62 [95.4%] with NDM-1) was the most common carbapenemase, followed by IMP (n= 7) and OXA (n= 7). Isolates from hospital-associated IAI had more complicated β-lactamase combinations than isolates from the community. Carbapenemases were all exclusively detected inEnterobacteriaceaeisolates from India, except that IMP β-lactamases were also detected in Philippines and Australia. CTX-M β-lactamases were the predominant ESBLs produced byEnterobacteriaceaecausing IAI in the Asia-Pacific region. Emergence of CTX-M-15-, CMY-2-, and NDM-1-producingEnterobacteriaceaeisolates is of major concern and highlights the need for further surveillance in this area.

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