As resistance determinants, KPC ␤-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type ␤-lactamases are often identified as resistant to standard ␤-lactam-␤-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to ␤-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (k cat /k inact ratios, where k inact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (k cat /k inact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (k inact /K m ‫؍‬ 6.5 ؎ 0.6 M ؊1 s ؊1 ). We conclude that KPC-2 is unique among class A ␤-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type ␤-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current ␤-lactamase inhibitors.In Klebsiella pneumoniae, ␤-lactam resistance is mediated predominantly by class A SHV, TEM, and CTX-M ␤-lactamases (7, 35). Single amino acid substitutions in the SHV and TEM ␤-lactamases can drastically alter the substrate profiles of the enzymes and confer resistance to extended-spectrum cephalosporins and ␤-lactamase inhibitors (5,12,34,36). ␤-Lactamases with altered substrate profiles (i.e., extended-spectrum or inhibitor-resistant ␤-lactamases) have significantly challenged the clinician's approach to the treatment of serious infectious diseases (36). Thus, the search for effective mechanism-based inhibitors of novel ␤-lactamases merits significant effort (8,9,32).First identified in K. pneumoniae, KPC class A ␤-lactamases threaten the use of all current ␤-lactam antibiotics (57). These ␤-lactamase enzymes are present in an increasing number of bacterial genera, becoming the major carbapenemase expressed by Gram-negative pathogens (e.g., Enterobacter spp., Escherichia coli, Citrobacter freundii, Pseudomonas spp., Serratia marcescens, Proteus mirabilis, and Salmonella enterica) in the United States (3,10,11,16,17,25,37,45,49,53,59). Moreover, KPC ␤-lactamases are becoming geographically widespread (having been detecte...

We demonstrated the complex beta-lactamase background of KPC-Kp isolates that are emerging in multiple centres in the Eastern USA. The prevalence of a single dominant clone suggests that interstate transmission has occurred.

Beta-lactamases inactivate beta-lactam antibiotics and are a major cause of antibiotic resistance. The recent outbreaks of Klebsiella pneumoniae carbapenem resistant (KPC) infections mediated by KPC type beta-lactamases are creating a serious threat to our "last resort" antibiotics, the carbapenems. KPC beta-lactamases are serine carbapenemases and are a subclass of class A beta-lactamases that have evolved to efficiently hydrolyze carbapenems and cephamycins which contain substitutions at the alpha-position proximal to the carbonyl group that normally render these beta-lactams resistant to hydrolysis. To investigate the molecular basis of this carbapenemase activity, we have determined the structure of KPC-2 at 1.85 A resolution. The active site of KPC-2 reveals the presence of a bicine buffer molecule which interacts via its carboxyl group with conserved active site residues S130, K234, T235, and T237; these likely resemble the interactions the beta-lactam carboxyl moiety makes in the Michaelis-Menten complex. Comparison of the KPC-2 structure with non-carbapenemases and previously determined NMC-A and SME-1 carbapenemase structures shows several active site alterations that are unique among carbapenemases. An outward shift of the catalytic S70 residue renders the active sites of the carbapenemases more shallow, likely allowing easier access of the bulkier substrates. Further space for the alpha-substituents is potentially provided by shifts in N132 and N170 in addition to concerted movements in the postulated carboxyl binding pocket that might allow the substrates to bind at a slightly different angle to accommodate these alpha-substituents. The structure of KPC-2 provides key insights into the carbapenemase activity of emerging class A beta-lactamases.

The efficacy of β-lactam antibiotics is threatened by the emergence and global spread of metallo-β-lactamase-(MBL) mediated resistance, specifically New Delhi-Metallo-β-lactamase-1 (NDM-1). Utilizing fragment-based drug discovery (FBDD), a new class of inhibitors for NDM-1 and two related β-lactamases, IMP-1 and VIM-2, was identified. Based on 2,6-dipicolinic acid (DPA), several libraries were synthesized for structure-activity relationship (SAR) analysis. Inhibitor 36 (IC50 = 80 nM) was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes. While DPA displayed a propensity to chelate metal ions from NDM-1, 36 formed a stable NDM-1:Zn(II):inhibitor ternary complex, as demonstrated by 1H NMR, electron paramagnetic resonance (EPR) spectroscopy, equilibrium dialysis, intrinsic tryptophan fluorescence emission, and UV-Vis spectroscopy. When co-administered with 36 (at concentrations non-toxic to mammalian cells), the minimum inhibitory concentration (MIC) of imipenem against clinical isolates of Eschericia coli and Klebsiella pneumoniae harboring NDM-1 were reduced to susceptible levels.

Limited treatment options exist to combat infections caused by multidrug-resistant (MDR) Gram-negative bacteria possessing broad-spectrum β-lactamases. The design of novel β-lactamase inhibitors is of paramount importance. Here, three novel diazabicyclooctanes (DBOs), WCK 5153, zidebactam (WCK 5107), and WCK 4234 (compounds 1-3, respectively), were synthesized and biochemically characterized against clinically important bacteria. Compound 3 inhibited class A, C, and D β-lactamases with unprecedented k/ K values against OXA carbapenemases. Compounds 1 and 2 acylated class A and C β-lactamses rapidly but not the tested OXAs. Compounds 1-3 formed highly stable acyl-complexes as demonstrated by mass spectrometry. Crystallography revealed that 1-3 complexed with KPC-2 adopted a "chair conformation" with the sulfate occupying the carboxylate binding region. The cefepime-2 and meropenem-3 combinations were effective in murine peritonitis and neutropenic lung infection models caused by MDR Acinetobacter baumannii. Compounds 1-3 are novel β-lactamase inhibitors that demonstate potent cross-class inhibition, and clinical studies targeting MDR infections are warranted.

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 g/ml) was identified. Herein, we sought to determine the molecular basis for the extended-spectrum-cephalosporin resistance. Using analytical isoelectric focusing, a ␤-lactamase with a pI of >9.2 was detected. PCR amplification with specific A. baumannii cephalosporinase primers yielded a 1,152-bp product which, when sequenced, identified a novel 383-amino-acid class C enzyme. Expressed in Escherichia coli DH10B, this ␤-lactamase demonstrated greater resistance against ceftazidime and cefotaxime than cefepime (4.0 g/ml versus 0.06 g/ml). The kinetic characteristics of this ␤-lactamase were similar to other cephalosporinases found in Acinetobacter spp. In addition, this cephalosporinase was inhibited by meropenem, imipenem, ertapenem, and sulopenem (K i < 40 M). The amino acid compositions of this novel enzyme and other class C ␤-lactamases thus far described for A. baumannii, Acinetobacter genomic species 3, and Oligella urethralis in Europe and South Africa suggest that this cephalosporinase defines a unique family of class C enzymes. We propose a uniform designation for this family of cephalosporinases (Acinetobacter-derived cephalosporinases [ADC]) found in Acinetobacter spp. and identify this enzyme as ADC-7 ␤-lactamase. The coalescence of Acinetobacter ampC ␤-lactamases into a single common ancestor and the substantial phylogenetic distance separating them from other ampC genes support the logical value of developing a system of nomenclature for these Acinetobacter cephalosporinase genes.Acinetobacter spp. are commonly associated with serious nosocomial infections (10,14,23,37,40,41). Most recently, American troops wounded in Iraq (Operation Iraqi Freedom) and Afghanistan (Operation Enduring Freedom) have suffered severe infections from antibiotic-resistant Acinetobacter baumannii, making this organism an important pathogen in military health

Carbapenem antibiotics are often the “last resort” in the treatment of infections caused by bacteria resistant to penicillins and cephalosporins. To understand why meropenem is resistant to hydrolysis by the SHV-1 class A β-lactamase, the atomic structure of meropenem inactivated SHV-1 was solved to 1.05 Å resolution. Two conformations of the Ser70 acylated intermediate are observed in the SHV-1-meropenem complex; the meropenem carbonyl oxygen atom of the acyl-enzyme is in the oxyanion hole in one conformation, while in the other conformation it is not. Although the structures of the SHV-1 apoenzyme and the SHV-1-meropenem complex are very similar (0.29 Å rmsd for Cα atoms), the orientation of the conserved Ser130 is different. Notably, the Ser130-OH group of the SHV-1-meropenem complex is directed toward Lys234Nz, while the Ser130-OH of the apo enzyme is oriented toward the Lys73 amino group. This altered position may affect proton transfer via Ser130 and the rate of hydrolysis. A most intriguing finding is the crystallographic detection of protonation of the Glu166 known to be involved in the deacylation mechanism. The critical deacylation water molecule has an additional hydrogen-bonding interaction with the OH group of meropenem’s 6α-1R-hydroxyethyl substituent. This interaction may weaken the nucleophilicity and/or change the direction of the lone pair of electrons of the water molecule and result in poor turnover of meropenem by the SHV-1 β-lactamase. Using timed mass spectrometry, we further show that meropenem is covalently attached to SHV-1 β-lactamase for at least 60 min. These observations explain key properties of meropenem’s ability to resist hydrolysis by SHV-1 and lead to important insights regarding future carbapenem and β-lactamase inhibitor design.

Infections by carbapenem-resistant Enterobacteriaceae (CRE) are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically-used β-lactamase inhibitors to counter the activity of metallo-β-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-β-lactamase (NDM), which requires a dinuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact of NDM variants on structure is probed by comparing comparing melting temperature and refolding efficiency and also by spectroscopy (UV-Vis, 1 H-NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining of minimum inhibitory concentrations of various antibiotics, pre-steady state and steadystate kinetics, inhibitor binding, and zincdependence of resistance and activity. We observed only minor differences among the fullloaded dizinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants http://www.jbc.org/ Downloaded from 2 exhibited improved thermostability (up to ~10 °C increased Tm) and improved zinc affinity (up to ~10-fold decreased Kd, Zn2). We also provide first evidence that some NDM variants have evolved the ability to function as monozinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/KM = 5 × 10 6 M -1 s -1 ). These findings reveal the molecular mechanisms that NDM variants have evolved to overcome the combined selective pressures of β-lactam antibiotics and zinc deprivation.Carbapenem-resistant Enterobacteriaceae (CRE) continue to be classified as an "urgent threat," the highest hazard level assigned by the Centers for Disease Control and Prevention(1). The five carbapenemases currently of primary public concern include Klebsiella pneumonia carbapanemase (KPC), New Delhi metallo-β-lactamase (NDM), Verona integrin encoded metallo-β-lactamase (VIM), imipenemase (IMP), and oxacillinase-48-like carbapenemase (OXA-48)(2). Three of these carbapenemases (NDM, VIM, and IMP) are metal-dependent β-lactamases that are not susceptible to any of the β-lactamase inhibitors incorporated into combination drugs used in the clinic. Of these three β-lactamases, NDM is the most widespread in U.S. patients, with infections bearing a blaNDM gene reported in 34 / 50 states (as of December 2017)(3).The genes encoding NDM continue to evolve, with discovery of more than 20 variants (NDM-1 through NDM-21 at the time of writing, 16 at the start of this project). Most of these mutations occur at sites distant from the active site, and the functions they confer are not immediately obvious. A comparison of NDM-1 through NDM-8 showed only minor differences in kcat/KM values (≤ 5-fold) for a panel of diverse β-lactam drugs(4). However, a considerable increase in thermostability was noted for many of the variants, suggesting the functional impact of NDM...