scholarly journals Cyclic Boronates Inhibit All Classes of β-Lactamases

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Samuel T. Cahill ◽  
Ricky Cain ◽  
David Y. Wang ◽  
Christopher T. Lohans ◽  
David W. Wareham ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Bacterial Infections ◽  
Binding Mode ◽  
Gram Negative ◽  
Content Type ◽  
Class A ◽  
Class D ◽  
Dual Action ◽  
The Common ◽  
Continued Use

ABSTRACT β-Lactamase-mediated resistance is a growing threat to the continued use of β-lactam antibiotics. The use of the β-lactam-based serine-β-lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non-β-lactam inhibitor avibactam has extended the utility of β-lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo-β-lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo-β-lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of “dual-action” inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difficult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo-β-lactamases. Here we report that cyclic boronates are able to inhibit all four classes of β-lactamase, including the class A extended spectrum β-lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa, and class D OXA enzymes with carbapenem-hydrolyzing capabilities. We demonstrate that cyclic boronates can potentiate the use of β-lactams against Gram-negative clinical isolates expressing a variety of β-lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other β-lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo-β-lactamase catalysis.

scholarly journals Avibactam and Inhibitor-Resistant SHV β-Lactamases

2015 ◽  
Vol 59 (7) ◽  
pp. 3700-3709 ◽  
Author(s):  
Marisa L. Winkler ◽  
Krisztina M. Papp-Wallace ◽  
Magdalena A. Taracila ◽  
Robert A. Bonomo
Keyword(s):  
Alternative Pathway ◽  
Proton Acceptor ◽  
Hydroxyl Group ◽  
The Novel ◽  
Content Type ◽  
Class A ◽  
Important Amino Acid ◽  
Class D ◽  
Continued Use ◽  
Lactamase Inhibitor

ABSTRACTβ-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase inhibitor with activity against many class A and C and some class D β-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of β-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a β-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter forblaSHV-1to 256 mg/liter forblaSHV S130Gexpressed inEscherichia coliDH10B. At steady state, thek2/Kvalue of the S130G variant when inactivated by avibactam was 1.3 M−1s−1, versus 60,300 M−1s−1for the SHV-1 β-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15–avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the β-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely also slow due to the lack of a general base to initiate the process. Considering other inhibitor-resistant mechanisms among class A β-lactamases, S130 may be the most important amino acid for the inhibition of class A β-lactamases, perhaps even for the novel diazabicyclooctane class of β-lactamase inhibitors.

scholarly journals Assessment of theIn VitroActivity of Ceftazidime-Avibactam against Multidrug-Resistant Klebsiella spp. Collected in the INFORM Global Surveillance Study, 2012 to 2014

2016 ◽  
Vol 60 (8) ◽  
pp. 4677-4683 ◽  
Author(s):  
Meredith Hackel ◽  
Krystyna M. Kazmierczak ◽  
Daryl J. Hoban ◽  
Douglas J. Biedenbach ◽  
Samuel K. Bouchillon ◽  
...  
Keyword(s):  
Multidrug Resistant ◽  
Surveillance Study ◽  
Gram Negative ◽  
Content Type ◽  
Class A ◽  
Highly Active ◽  
Class D ◽  
Class B ◽  
Klebsiella Spp

ABSTRACTIncreasing resistance in Gram-negative bacilli, includingKlebsiellaspp., has reduced the utility of broad-spectrum cephalosporins. Avibactam, a novel non-β-lactam β-lactamase inhibitor, protects β-lactams from hydrolysis by Gram-negative bacteria that produce extended-spectrum β-lactamases (ESBLs) and serine carbapenemases, including Ambler class A and/or class C and some class D enzymes. In this analysis, we report thein vitroactivity of ceftazidime-avibactam and comparators against multidrug-resistant (MDR)Klebsiellaspp. from the 2012-2014 INFORM surveillance study. Isolates collected from 176 sites were sent to a central laboratory for confirmatory identification and tested for susceptibility to ceftazidime-avibactam and comparator agents, including ceftazidime alone. A total of 2,821 of 10,998 (25.7%)Klebsiellaspecies isolates were classified as MDR, based on resistance to three or more classes of antimicrobials. Among the MDR isolates, 99.4% had an ESBL screen-positive phenotype, and 27.4% were not susceptible to meropenem as an example of a carbapenem. Ceftazidime-avibactam was highly active against MDR isolates, including ESBL-positive and serine carbapenemase-producing isolates, with MIC50/90values of 0.5/2 μg/ml and 96.6% of all MDR isolates and ESBL-positive MDR isolates inhibited at the FDA breakpoint (MIC value of ≤8 μg/ml). Ceftazidime-avibactam did not inhibit isolates producing class B enzymes (metallo-β-lactamases) either alone or in combination with other enzymes. Thesein vitroresults support the continued investigation of ceftazidime-avibactam for the treatment of MDRKlebsiellaspecies infections.

scholarly journals Genetic Diversity, Biochemical Properties, and Detection Methods of Minor Carbapenemases in Enterobacterales

2021 ◽  
Vol 7 ◽  
Author(s):  
Rémy A. Bonnin ◽  
Agnès B. Jousset ◽  
Cécile Emeraud ◽  
Saoussen Oueslati ◽  
Laurent Dortet ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Multidrug Resistant ◽  
Biochemical Properties ◽  
Amino Acid Sequences ◽  
Detection Methods ◽  
Gram Negative ◽  
Class A ◽  
Class D ◽  
Class B ◽  
Encoding Genes

Gram-negative bacteria, especially Enterobacterales, have emerged as major players in antimicrobial resistance worldwide. Resistance may affect all major classes of anti-gram-negative agents, becoming multidrug resistant or even pan-drug resistant. Currently, β-lactamase-mediated resistance does not spare even the most powerful β-lactams (carbapenems), whose activity is challenged by carbapenemases. The dissemination of carbapenemases-encoding genes among Enterobacterales is a matter of concern, given the importance of carbapenems to treat nosocomial infections. Based on their amino acid sequences, carbapenemases are grouped into three major classes. Classes A and D use an active-site serine to catalyze hydrolysis, while class B (MBLs) require one or two zinc ions for their activity. The most important and clinically relevant carbapenemases are KPC, IMP/VIM/NDM, and OXA-48. However, several carbapenemases belonging to the different classes are less frequently detected. They correspond to class A (SME-, Nmc-A/IMI-, SFC-, GES-, BIC-like…), to class B (GIM, TMB, LMB…), class C (CMY-10 and ACT-28), and to class D (OXA-372). This review will address the genetic diversity, biochemical properties, and detection methods of minor acquired carbapenemases in Enterobacterales.

scholarly journals Whole-Animal Chemical Screen Identifies Colistin as a New Immunomodulator That Targets Conserved Pathways

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Yun Cai ◽  
Xiou Cao ◽  
Alejandro Aballay
Keyword(s):  
Transcription Factor ◽  
Immune Responses ◽  
Immune Activation ◽  
Bacterial Infections ◽  
Mitogen Activated Protein Kinase ◽  
Resistant Bacteria ◽  
Long Term Outcome ◽  
Gram Negative ◽  
Content Type ◽  
Chemical Screen

ABSTRACTThe purpose of this study was to take advantage of the nematodeCaenorhabditis elegansto perform a whole-animal chemical screen to identify potential immune activators that may confer protection against bacterial infections. We identified 45 marketed drugs, out of 1,120 studied compounds, that are capable of activating a conserved p38/PMK-1 mitogen-activated protein kinase pathway required for innate immunity. One of these drugs, the last-resort antibiotic colistin, protected against infections by the Gram-negative pathogensYersinia pestisandPseudomonas aeruginosabut not by the Gram-positive pathogensEnterococcus faecalisandStaphylococcus aureus. Protection was independent of the antibacterial activity of colistin, since the drug was administered prophylactically prior to the infections and it was also effective against antibiotic-resistant bacteria. Immune activation by colistin is mediated not only by the p38/PMK-1 pathway but also by the conserved FOXO transcription factor DAF-16 and the transcription factor SKN-1. Furthermore, p38/PMK-1 was found to be required in the intestine for immune activation by colistin. Enhanced p38/PMK-1-mediated immune responses by colistin did not reduce the bacterial burden, indicating that the pathway plays a role in the development of host tolerance to infections by Gram-negative bacteria.IMPORTANCEThe innate immune system represents the front line of our defenses against invading microorganisms. Given the ever-increasing resistance to antibiotics developed by bacterial pathogens, the possibility of boosting immune defenses represents an interesting, complementary approach to conventional antibiotic treatments. Here we report that the antibiotic colistin can protect against infections by a mechanism that is independent of its microbicidal activity. Prophylactic treatment with colistin activates a conserved p38/PMK-1 pathway in the intestine that helps the host better tolerate a bacterial infection. Since p38/PMK-1-mediated immune responses appear to be conserved from plants to mammals, colistin may also activate immunity in higher organisms, including humans. Antibiotics with immunomodulatory properties have the potential of improving the long-term outcome of patients with chronic infectious diseases.

scholarly journals Dynamics of Chromosome Replication and Its Relationship to Predatory Attack Lifestyles inBdellovibrio bacteriovorus

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
Łukasz Makowski ◽  
Damian Trojanowski ◽  
Rob Till ◽  
Carey Lambert ◽  
Rebecca Lowry ◽  
...  
Keyword(s):  
Life Cycle ◽  
Subcellular Localization ◽  
Bacterial Infections ◽  
Chromosome Replication ◽  
Free Living ◽  
Gram Negative ◽  
Content Type ◽  
Reproductive Phase ◽  
Attack Phase ◽  
Two Phases

ABSTRACTBdellovibrio bacteriovorusis a small Gram-negative, obligate predatory bacterium that is largely found in wet, aerobic environments (e.g., soil). This bacterium attacks and invades other Gram-negative bacteria, including animal and plant pathogens. The intriguing life cycle ofB. bacteriovorusconsists of two phases: a free-living nonreplicative attack phase, in which the predatory bacterium searches for its prey, and a reproductive phase, in whichB. bacteriovorusdegrades a host’s macromolecules and reuses them for its own growth and chromosome replication. Although the cell biology of this predatory bacterium has gained considerable interest in recent years, we know almost nothing about the dynamics of its chromosome replication. Here, we performed a real-time investigation into the subcellular localization of the replisome(s) in single cells ofB. bacteriovorus. Our results show that inB. bacteriovorus, chromosome replication takes place only during the reproductive phase and exhibits a novel spatiotemporal arrangement of replisomes. The replication process starts at the invasive pole of the predatory bacterium inside the prey cell and proceeds until several copies of the chromosome have been completely synthesized. Chromosome replication is not coincident with the predator cell division, and it terminates shortly before synchronous predator filament septation occurs. In addition, we demonstrate that if thisB. bacteriovoruslife cycle fails in some cells ofEscherichia coli, they can instead use second prey cells to complete their life cycle.IMPORTANCENew strategies are needed to combat multidrug-resistant bacterial infections. Application of the predatory bacteriumBdellovibrio bacteriovorus, which kills other bacteria, including pathogens, is considered promising for combating bacterial infections. TheB. bacteriovoruslife cycle consists of two phases, a free-living, invasive attack phase and an intracellular reproductive phase, in which this predatory bacterium degrades the host’s macromolecules and reuses them for its own growth. To understand the use ofB. bacteriovorusas a “living antibiotic,” it is first necessary to dissect its life cycle, including chromosome replication. Here, we present a real-time investigation into subcellular localization of chromosome replication in a single cell ofB. bacteriovorus. This process initiates at the invasion pole ofB. bacteriovorusand proceeds until several copies of the chromosome have been completely synthesized. Interestingly, we demonstrate that some cells ofB. bacteriovorusrequire two prey cells sequentially to complete their life cycle.

scholarly journals Active-Site Plasticity Is Essential to Carbapenem Hydrolysis by OXA-58 Class D β-Lactamase of Acinetobacter baumannii

2015 ◽  
Vol 60 (1) ◽  
pp. 75-86 ◽  
Author(s):  
Shivendra Pratap ◽  
Madhusudhanarao Katiki ◽  
Preet Gill ◽  
Pravindra Kumar ◽  
Dasantila Golemi-Kotra
Keyword(s):  
Crystal Structure ◽  
Acinetobacter Baumannii ◽  
Active Site ◽  
Active Sites ◽  
Multidrug Resistant ◽  
Crystal Structure Analysis ◽  
Content Type ◽  
Class D ◽  
Enzyme Species ◽  
Hydroxyethyl Group

ABSTRACTCarbapenem-hydrolyzing class D β-lactamases (CHDLs) are a subgroup of class D β-lactamases, which are enzymes that hydrolyze β-lactams. They have attracted interest due to the emergence of multidrug-resistantAcinetobacter baumannii, which is not responsive to treatment with carbapenems, the usual antibiotics of choice for this bacterium. Unlike other class D β-lactamases, these enzymes efficiently hydrolyze carbapenem antibiotics. To explore the structural requirements for the catalysis of carbapenems by these enzymes, we determined the crystal structure of the OXA-58 CHDL ofA. baumanniifollowing acylation of its active-site serine by a 6α-hydroxymethyl penicillin derivative that is a structural mimetic for a carbapenem. In addition, several point mutation variants of the active site of OXA-58, as identified by the crystal structure analysis, were characterized kinetically. These combined studies confirm the mechanistic relevance of a hydrophobic bridge formed over the active site. This structural feature is suggested to stabilize the hydrolysis-productive acyl-enzyme species formed from the carbapenem substrates of this enzyme. Furthermore, our structural studies provide strong evidence that the hydroxyethyl group of carbapenems samples different orientations in the active sites of CHDLs, and the optimum orientation for catalysis depends on the topology of the active site allowing proper closure of the active site. We propose that CHDLs use the plasticity of the active site to drive the mechanism of carbapenem hydrolysis toward efficiency.

scholarly journals Interactions of Ceftobiprole with β-Lactamases from Molecular Classes A to D

2007 ◽  
Vol 51 (9) ◽  
pp. 3089-3095 ◽  
Author(s):  
Anne Marie Queenan ◽  
Wenchi Shang ◽  
Malgosia Kania ◽  
Malcolm G. P. Page ◽  
Karen Bush
Keyword(s):  
Functional Groups ◽  
Hydrolytic Stability ◽  
Enterobacter Cloacae ◽  
Class A ◽  
Extended Spectrum ◽  
Class D ◽  
The Family ◽  
Class B ◽  
The Common ◽  
Antibacterial Spectrum

ABSTRACT The interactions of ceftobiprole with purified β-lactamases from molecular classes A, B, C, and D were determined and compared with those of benzylpenicillin, cephaloridine, cefepime, and ceftazidime. Enzymes were selected from functional groups 1, 2a, 2b, 2be, 2d, 2e, and 3 to represent β-lactamases from organisms within the antibacterial spectrum of ceftobiprole. Ceftobiprole was refractory to hydrolysis by the common staphylococcal PC1 β-lactamase, the class A TEM-1 β-lactamase, and the class C AmpC β-lactamase but was labile to hydrolysis by class B, class D, and class A extended-spectrum β-lactamases. Cefepime and ceftazidime followed similar patterns. In most cases, the hydrolytic stability of a substrate correlated with the MIC for the producing organism. Ceftobiprole and cefepime generally had lower MICs than ceftazidime for AmpC-producing organisms, particularly AmpC-overexpressing Enterobacter cloacae organisms. However, all three cephalosporins were hydrolyzed very slowly by AmpC cephalosporinases, suggesting that factors other than β-lactamase stability contribute to lower ceftobiprole and cefepime MICs against many members of the family Enterobacteriaceae.

scholarly journals Cryo-electron Microscopy Structure of the Acinetobacter baumannii 70S Ribosome and Implications for New Antibiotic Development

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Christopher E. Morgan ◽  
Wei Huang ◽  
Susan D. Rudin ◽  
Derek J. Taylor ◽  
James E. Kirby ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Acinetobacter Baumannii ◽  
Bacterial Infections ◽  
Bound States ◽  
Antimicrobial Agents ◽  
Gram Negative ◽  
Content Type ◽  
Antibiotic Development ◽  
Cryo Electron Microscopy ◽  
70S Ribosome

ABSTRACT Antimicrobial resistance is a major health threat as it limits treatment options for infection. At the forefront of this serious issue is Acinetobacter baumannii, a Gram-negative opportunistic pathogen that exhibits the remarkable ability to resist antibiotics through multiple mechanisms. As bacterial ribosomes represent a target for multiple distinct classes of existing antimicrobial agents, we here use single-particle cryo-electron microscopy (cryo-EM) to elucidate five different structural states of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. We also determined interparticle motions of the 70S ribosome in different tRNA bound states using three-dimensional (3D) variability analysis. Together, our structural data further our understanding of the ribosome from A. baumannii and other Gram-negative pathogens and will enable structure-based drug discovery to combat antibiotic-resistant bacterial infections. IMPORTANCE Acinetobacter baumannii is a severe nosocomial threat largely due to its intrinsic antibiotic resistance and remarkable ability to acquire new resistance determinants. The bacterial ribosome serves as a major target for modern antibiotics and the design of new therapeutics. Here, we present cryo-EM structures of the A. baumannii 70S ribosome, revealing several unique species-specific structural features that may facilitate future drug development to combat this recalcitrant bacterial pathogen.

scholarly journals Structural Basis and Binding Kinetics of Vaborbactam in Class A β-Lactamase Inhibition

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Orville A. Pemberton ◽  
Ruslan Tsivkovski ◽  
Maxim Totrov ◽  
Olga Lomovskaya ◽  
Yu Chen
Keyword(s):  
Crystal Structure ◽  
Covalent Bond ◽  
Binding Mode ◽  
Binding Kinetics ◽  
Structural Basis ◽  
Inhibition Mechanism ◽  
Class A ◽  
Reversible Covalent ◽  
The Stability ◽  
M 14

ABSTRACT Class A β-lactamases are a major cause of β-lactam resistance in Gram-negative bacteria. The recently FDA-approved cyclic boronate vaborbactam is a reversible covalent inhibitor of class A β-lactamases, including CTX-M extended-spectrum β-lactamase and KPC carbapenemase, both frequently observed in the clinic. Intriguingly, vaborbactam displayed different binding kinetics and cell-based activity for these two enzymes, despite their similarity. A 1.0-Å crystal structure of CTX-M-14 demonstrated that two catalytic residues, K73 and E166, are positively charged and neutral, respectively. Meanwhile, a 1.25-Å crystal structure of KPC-2 revealed a more compact binding mode of vaborbactam versus CTX-M-14, as well as alternative conformations of W105. Together with kinetic analysis of W105 mutants, the structures demonstrate the influence of this residue and the unusual conformation of the β3 strand on the inactivation rate, as well as the stability of the reversible covalent bond with S70. Furthermore, studies of KPC-2 S130G mutant shed light on the different impacts of S130 in the binding of vaborbactam versus avibactam, another recently approved β-lactamase inhibitor. Taken together, these new data provide valuable insights into the inhibition mechanism of vaborbactam and future development of cyclic boronate inhibitors.

scholarly journals Urinary Concentrations of Colistimethate and Formed Colistin after Intravenous Administration in Patients with Multidrug-Resistant Gram-Negative Bacterial Infections

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Sonia Luque ◽  
Carol Escaño ◽  
Luisa Sorli ◽  
Jian Li ◽  
Nuria Campillo ◽  
...  
Keyword(s):  
Urinary Excretion ◽  
Intravenous Administration ◽  
Bacterial Infections ◽  
High Performance ◽  
Multidrug Resistant ◽  
Limited Information ◽  
Urinary Recovery ◽  
Gram Negative ◽  
Content Type ◽  
Colistimethate Sodium

ABSTRACT Limited information is available on the urinary excretion of colistin in infected patients. This study aimed to investigate the pharmacokinetics of colistimethate sodium (CMS) and formed colistin in urine in patients with multidrug-resistant (MDR) Gram-negative bacterial infections. A pharmacokinetic study was conducted on 12 patients diagnosed with an infection caused by an extremely drug-resistant (XDR) P. aeruginosa strain and treated with intravenous CMS. Fresh urine samples were collected at 2-h intervals, and blood samples were collected predose (C min ss) and at the end of the CMS infusion (C max ss) for measurement of concentrations of CMS and formed colistin using high-performance liquid chromatography (HPLC). CMS urinary recovery was determined as the summed amount of CMS and formed colistin recovered in urine for each 2-h interval divided by the CMS dose. There were 12 enrolled patients, 9 of whom were male (75%). Data [median (range)] were as follows: age, 65.5 (37 to 86) years; colistimethate urinary recovery 0 to 6 h, 42.6% (2.9% to 72.8%); range of concentrations of colistin in urine, <0.1 to 95.4 mg/liter; C min ss and C max ss of colistin in plasma, 0.9 (<0.2 to 1.4) and 0.9 (<0.2 to 1.4) mg/liter, respectively. In 6/12 (50%) patients, more than 40% of the CMS dose was recovered in the urine within the first 6 h after CMS administration. This study demonstrated rapid urinary excretion of CMS in patients within the first 6 h after intravenous administration. In all but one patient, the concentrations of formed colistin in urine were above the MIC for the most predominant isolate of P. aeruginosa in our hospital. Future studies are warranted for optimizing CMS dosage regimens in urinary tract infection (UTI) patients.

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