Ab Initio Raman Spectra of β-Lactamase Inhibitor Intermediates Bound to E166A SHV β-Lactamase.

The assignment and the analysis of the experimental vibrational Raman spectra of enzyme bound β-lactamase inhibitors may be of help to understand the mechanisms responsible for bacterial drug resistance. We present a computational study of the structural and vibrational properties of clavulanic acid and tazobactam intermediates, two important β-lactamase inhibitors, bound to the singly mutated E166A SHV β-lactamase in aqueous solution by hybrid molecular mechanics/quantum mechanics (QM/MM) simulations at ambient conditions. We compare the Raman spectra obtained from the time autocorrelation function of polarizability tensor as obtained from a QM/MM protocol to those obtained from the instantaneous normal modes analysis performed on top of the QM/MM trajectory in order to establish the accuracy of these two computational methods and to review the previously made assignments.

Inhibition of OXA-1 beta-lactamase by penems.

The partnering of a beta-lactam with a beta-lactamase inhibitor is a highly effective strategy that can be used to combat bacterial resistance to beta-lactam antibiotics mediated by serine beta-lactamases (EC 3.2.5.

Sulbactam forms only minimal amounts of irreversible acrylate-enzyme with SHV-1 beta-lactamase.

Sulbactam is a mechanism-based inhibitor of beta-lactamase enzymes used in clinical practice. It undergoes a complex series of chemical reactions in the active site that have been studied extensively in the past three decades. However, the actual species that gives rise to inhibition in a clinical setting has not been established.

Raman crystallographic studies of the intermediates formed by Ser130Gly SHV, a beta-lactamase that confers resistance to clinical inhibitors.

Antibiotic resistance to beta-lactam compounds in Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae is often mediated by beta-lactamase enzymes like TEM and SHV. Previously, a limited number of inhibitors have shown efficacy in combating such bacterial drug resistance. However, many Gram-negative pathogens have evolved inhibitor resistant forms of these hydrolytic enzymes.

Structure and Raman spectrum of clavulanic acid in aqueous solution.

The calculation of the vibrational Raman spectrum of enzyme-bound beta-lactamase inhibitors may be of help to understand the mechanisms responsible for bacterial drug resistance. Here, we present a study of the solvation structure and the vibrational properties of clavulanate, an important beta-lactamase inhibitor, in aqueous solution as obtained from full quantum and hybrid empirical/quantum molecular dynamics simulations at ambient conditions. The analysis of the vibrational density of states indicates that hybrid empirical/quantum mechanical simulations are able to properly describe the vibrational levels of clavulanate in solution.

Rational design of a beta-lactamase inhibitor achieved via stabilization of the trans-enamine intermediate: 1.28 A crystal structure of wt SHV-1 complex with a penam sulfone.

beta-Lactamases are one of the major causes of antibiotic resistance in Gram negative bacteria. The continuing evolution of beta-lactamases that are capable of hydrolyzing our most potent beta-lactams presents a vexing clinical problem, in particular since a number of them are resistant to inhibitors. The efficient inhibition of these enzymes is therefore of great clinical importance.

Effect of the inhibitor-resistant M69V substitution on the structures and populations of trans-enamine beta-lactamase intermediates.

The objective of this study was to determine the molecular factors that lead to beta-lactamase inhibitor resistance for the M69V variant in SHV-1 beta-lactamase. With mechanism-based inhibitors, the beta-lactamase forms an acyl-enzyme intermediate that consists of a trans-enamine derivative in the active site. This study focuses on these intermediates by introducing the E166A mutation that greatly retards deacylation.

Role of Asp104 in the SHV beta-lactamase.

Among the TEM-type extended-spectrum beta-lactamases (ESBLs), an amino acid change at Ambler position 104 (Glu to Lys) results in increased resistance to ceftazidime and cefotaxime when found with other substitutions (e.g., Gly238Ser and Arg164Ser).

Current challenges in antimicrobial chemotherapy: the impact of extended-spectrum beta-lactamases and metallo-beta-lactamases on the treatment of resistant Gram-negative pathogens.

Gram-negative bacteria that produce extended-spectrum- and metallo-beta-lactamases are being discovered at an alarming rate, while the development of new antimicrobial agents has almost ground to a standstill. A body of experience exists with the detection and treatment of extended-spectrum beta-lactamase-producing organisms (Klebsiella pneumoniae and Escherichia coli), suggesting that knowledge of their existence and dissemination might have an impact on therapeutic choices and patient outcomes via targeted empirical antimicrobial selection and infection control practices. It is unclear whether the same mandate exists for the detection of metallo-beta-lactamases.

High resolution crystal structures of the trans-enamine intermediates formed by sulbactam and clavulanic acid and E166A SHV-1 {beta}-lactamase.

Antibiotic resistance mediated by constantly evolving beta-lactamases is a serious threat to human health. The mechanism of inhibition of these enzymes by therapeutic beta-lactamase inhibitors is probed using a novel approach involving Raman microscopy and x-ray crystallography. We have presented here the high resolution crystal structures of the beta-lactamase inhibitors sulbactam and clavulanic acid bound to the deacylation-deficient E166A variant of SHV-1 beta-lactamase.

Clavulanic acid inactivation of SHV-1 and the inhibitor-resistant S130G SHV-1 beta-lactamase. Insights into the mechanism of inhibition.

Clavulanic acid is a potent mechanism-based inhibitor of TEM-1 and SHV-1beta-lactamases, enzymes that confer resistance to beta-lactams in many gram-negative pathogens. This compound has enjoyed widespread clinical use as part of beta-lactam beta-lactamase inhibitor therapy directed against penicillin-resistant pathogens. Unfortunately, the emergence of clavulanic acid-resistant variants of TEM-1 and SHV-1 beta-lactamase significantly compromise the efficacy of this combination.

Identification of a new allelic variant of the Acinetobacter baumannii cephalosporinase, ADC-7 beta-lactamase: defining a unique family of class C enzymes.

Acinetobacter spp. are emerging as opportunistic hospital pathogens that demonstrate resistance to many classes of antibiotics. In a metropolitan hospital in Cleveland, a clinical isolate of Acinetobacter baumannii that tested resistant to cefepime and ceftazidime (MIC = 32 microg/ml) was identified.

Mechanisms by which anaerobic microbiota inhibit the establishment in mice of intestinal colonization by vancomycin-resistant Enterococcus.

We used a mouse model to test the hypothesis that anaerobic microbiota in the colon inhibit the establishment of vancomycin-resistant enterococci (VRE) colonization by depleting nutrients within cecal contents and limiting the association of VRE with the mucus layer. Anaerobic growth of VRE was assessed in cecal contents and cecal mucus of mice that had received treatment with subcutaneous clindamycin or saline. VRE grew to high concentrations in cecal contents of clindamycin-treated mice and in cecal mucus of both groups but not in cecal contents of saline-treated mice, unless the cecal contents were autoclaved or converted into sterile filtrates.

Effect of parenteral antibiotic administration on establishment of intestinal colonization by Candida glabrata in adult mice.

We examined the effect of antibiotic treatment on establishment of intestinal colonization by Candida glabrata in adult mice. Subcutaneous ceftriaxone, piperacillin-tazobactam, clindamycin, and metronidazole promoted increased density of stool colonization, whereas cefepime, levofloxacin, and aztreonam did not. These findings suggest that antibiotics that inhibit intestinal anaerobes promote C.

In vitro antienterococcal activity explains associations between exposures to antimicrobial agents and risk of colonization by multiresistant enterococci.

We compared ceftriaxone and piperacillin-tazobactam at doses ranging from 0.1 to 2 times the human equivalent daily dose (HEDD), to determine their impact on gastrointestinal colonization by ampicillin- and vancomycin-resistant Enterococcus faecium C68 in a mouse model. Ceftriaxone failed to promote colonization at doses up to 0.

Increased susceptibility to vancomycin-resistant Enterococcus intestinal colonization persists after completion of anti-anaerobic antibiotic treatment in mice.

Background: Antibiotic-associated disruption of the indigenous intestinal microflora may persist beyond the treatment period. Although piperacillin/tazobactam inhibits the establishment of vancomycin-resistant Enterococcus (VRE) stool colonization in mice during treatment, we hypothesized that this agent and other anti-anaerobic antibiotics would increase susceptibility to colonization during the period of recovery of the intestinal microflora.

Design: Mice received 10(4) colony-forming units of vancomycin-resistant E.

Efficacy of oral ramoplanin for inhibition of intestinal colonization by vancomycin-resistant enterococci in mice.

Ramoplanin is a glycolipodepsipeptide antibiotic with activity against gram-positive bacteria that is in clinical trials for prevention of vancomycin-resistant Enterococcus (VRE) bloodstream infections and treatment of Clostridium difficile diarrhea. Orally administered ramoplanin suppresses VRE intestinal colonization, but recurrences after discontinuation of treatment have frequently been observed. We used a mouse model to examine the efficacy of ramoplanin for inhibition of VRE colonization and evaluated the etiology of recurrences of colonization.

Exploring the effectiveness of tazobactam against ceftazidime resistant Escherichia coli: insights from the comparison between susceptibility testing and beta-lactamase inhibition.

Thirteen clinical isolates of Escherichia coli resistant to ceftazidime that possessed an AmpC and other (beta-lactamases were identified. The effectiveness of different formulations of piperacillin/tazobactam to other beta-lactams was compared. Antibiotic susceptibility testing, polymerase chain reaction, amplification of blaTEM, blaSHV and blaAmpC, and enzyme-linked immunosorbent assays to identify AmpC beta-lactamases were performed.

Beta-lactam antibiotics and gastrointestinal colonization with vancomycin-resistant enterococci.

We studied the effect of different subcutaneously administered beta-lactam antibiotics on the establishment of gastrointestinal colonization by vancomycin-resistant Enterococcus faecium C68 in a mouse model. Aztreonam, cefazolin, cefepime, and, to a lesser extent, ceftazidime, which neither have significant antienterococcal activity nor are secreted into human bile at high concentrations, did not promote significant vancomycin-resistant enterococci (VRE) colonization. Piperacillin-tazobactam, which has antienterococcal activity and is secreted in human bile at high concentrations, inhibited colonization after limited exposure to the inoculum but was associated with progressively increased VRE colony counts in stool samples after repeated exposure to the VRE inoculum.

Tazobactam inactivation of SHV-1 and the inhibitor-resistant Ser130 -->Gly SHV-1 beta-lactamase: insights into the mechanism of inhibition.

The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor.

Tazobactam forms a stoichiometric trans-enamine intermediate in the E166A variant of SHV-1 beta-lactamase: 1.63 A crystal structure.

Many pathogenic bacteria develop antibiotic resistance by utilizing beta-lactamases to degrade penicillin-like antibiotics. A commonly prescribed mechanism-based inhibitor of beta-lactamases is tazobactam, which can function either irreversibly or in a transient manner. We have demonstrated previously that the reaction between tazobactam and a deacylation deficient variant of SHV-1 beta-lactamase, E166A, could be followed in single crystals using Raman microscopy [Helfand, M.

Effect of parenteral fluoroquinolone administration on persistence of vancomycin-resistant Enterococcus faecium in the mouse gastrointestinal tract.

We examined the effect of subcutaneous fluoroquinolone antibiotic administration on persistence and density of vancomycin-resistant Enterococcus faecium stool colonization in mice. Levofloxacin and ciprofloxacin did not promote colonization in comparison to saline controls, whereas moxifloxacin and gatifloxacin promoted persistent overgrowth in a dose-dependent fashion.

Oral administration of beta-lactamase preserves colonization resistance of piperacillin-treated mice.

We hypothesized that orally administered, recombinant class A beta-lactamase would inactivate the portion of parenteral piperacillin excreted into the intestinal tract, preserving colonization resistance of mice against nosocomial pathogens. Subcutaneous piperacillin or piperacillin plus oral beta-lactamase were administered 24 and 12 h before orogastric inoculation of piperacillin-resistant pathogens. Oral administration of beta-lactamase reduced piperacillin-associated alteration of the indigenous microflora and prevented overgrowth of pathogens.