Restricted Rotational Flexibility of the C5α-Methyl-Substituted Carbapenem NA-1-157 Leads to Potent Inhibition of the GES-5 Carbapenemase.

Carbapenem antibiotics are used as a last-resort treatment for infections caused by multidrug-resistant bacteria. The wide spread of carbapenemases in Gram-negative bacteria has severely compromised the utility of these drugs and represents a serious public health threat. To combat carbapenemase-mediated resistance, new antimicrobials and inhibitors of these enzymes are urgently needed. Here, we describe the interaction of the atypically C5α-methyl-substituted carbapenem, NA-1-157, with the GES-5 carbapenemase. MICs of this compound against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii producing the enzyme were reduced 4-16-fold when compared to MICs of the commercial carbapenems, reaching clinically sensitive breakpoints. When NA-1-157 was combined with meropenem, a strong synergistic effect was observed. Kinetic and ESI-LC/MS studies demonstrated that NA-1-157 is a potent inhibitor of GES-5, with a high inactivation efficiency of (2.9 ± 0.9) × 105 M-1 s-1. Acylation of GES-5 by NA-1-157 was biphasic, with the fast phase completing within seconds, and the slow phase taking several hours and likely proceeding through a reversible tetrahedral intermediate. Deacylation was extremely slow (k3 = (2.4 ± 0.3) × 10-7 s-1), resulting in a residence time of 48 ± 6 days. MD simulation of the GES-5-meropenem and GES-5-NA-1-157 acyl-enzyme complexes revealed that the C5α-methyl group in NA-1-157 sterically restricts rotation of the 6α-hydroxyethyl group preventing ingress of the deacylating water into the vicinity of the scissile bond of the acyl-enzyme intermediate. These data demonstrate that NA-1-157 is a potent irreversible inhibitor of the GES-5 carbapenemase.

Novel metallo-ß-lactamases inhibitors restore the susceptibility of carbapenems to New Delhi metallo-lactamase-1 (NDM-1)-harbouring bacteria.

BACKGROUND AND PURPOSE: The production of metallo-ß-lactamases is a major mechanisms adopted by bacterial pathogens to resist carbapenems. Repurposing approved drugs to restore the efficacy of carbapenems represents an efficient and cost-effective approach to fight infections caused by carbapenem resistant pathogens. EXPERIMENTAL APPROACH: The nitrocefin hydrolysis assay was employed to screen potential New Delhi metallo-lactamase-1 (NDM-1) inhibitors from a commercially available U.S. Food and Drug Administration (FDA) approved drug library. The mechanism of inhibition was clarified by metal restoration, inductively coupled plasma mass spectrometry (ICP-MS) and molecular dynamics simulation. The in vitro synergistic antibacterial effect of the identified inhibitors with meropenem was determined by the checkerboard minimum inhibitory concentration (MIC) assay, time-dependent killing assay and combined disc test. Three mouse infection models were used to further evaluate the in vivo therapeutic efficacy of combined therapy. KEY RESULTS: Twelve FDA-approved compounds were initially screened to inhibit the ability of NDM-1 to hydrolyse nitrocefin. Among these compounds, dexrazoxane, embelin, candesartan cilexetil and nordihydroguaiaretic acid were demonstrated to inhibit all tested metallo-ß-lactamases and showed an in vitro synergistic bactericidal effect with meropenem against metallo-ß-lactamases-producing bacteria. Dexrazoxane, embelin and candesartan cilexetil are metal ion chelating agents, while the inhibition of NDM-1 by nordihydroguaiaretic acid involves its direct binding to the active region of NDM-1. Furthermore, these four drugs dramatically rescued the treatment efficacy of meropenem in three infection models. CONCLUSIONS AND IMPLICATIONS: Our observations indicated that dexrazoxane, embelin, candesartan cilexetil and nordihydroguaiaretic acid are promising carbapenem adjuvants against metallo-ß-lactamases-positive carbapenem resistant bacterial pathogens.

Metallo-ß-lactamase SMB-1 evolves into a more efficient hydrolase under the selective pressure of meropenem.

Metallo-ß-lactamases (MßLs) are the primary mechanism of resistance to carbapenem antibiotics. To elucidate how MßLs have evolved with the introduction and use of antibiotics, the mutation and evolution of SMB-1 from Serratia marcescens were investigated in microbial evolution plates containing discontinuous meropenem (MEM) concentration gradients. The results revealed 2-point mutations, A242G and S257R; 1 double-site mutation, C240G/E258G; and 3 frameshift mutations, M5, M12, and M13, which are all missense mutations situated at the C-terminus. Compared with that of the wild-type (WT), the minimum inhibitory concentrations (MICs) of MEM for A242G, C240G/E258G, M5, M12, and M13 increased at least 120-fold, and that of S257R increased 8-fold. The catalytic efficiency kcat/Km increased by 365% and 647%, respectively. Concerning the structural changes, the structure at the active site changed from an ordered structure to an unordered conformation. Simultaneously, the flexibility of loop 1 was enhanced. These changes increased the volume of the active site cavity; thus, this was more conducive to exposing the Zn2+ site, facilitating substrate binding and conversion to products. In A242G, structural changes in Gly-242 can be transmitted to the active region via a network of interactions between the side chains of Gly-242 and the amino acid side chains near the active pocket. Together, these results pointed to the process of persistent drug tolerance and resistance, the SMB-1 enzyme evolved into a more exquisite structure with increased flexibility and stability, and stronger hydrolysis activity via genetic mutations and structural changes.

Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 ß-Lactamase.

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-ß-lactamase (SBL) responsible for extensive ß-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate ß-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent ß-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25-1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different ß-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.

The Effects of One-Point Mutation on the New Delhi Metallo Beta-Lactamase-1 Resistance toward Carbapenem Antibiotics and ß-Lactamase Inhibitors: An In Silico Systematic Approach.

Antibiotic resistance has been becoming more and more critical due to bacteria's evolving hydrolysis enzymes. The NDM-1 enzyme could hydrolyze not only carbapenems but also most of ß-lactam's antibiotics and inhibitors. In fact, variant strains could impose a high impact on the resistance of bacteria producing NDM-1. Although previous studies showed the effect of some variants toward antibiotics and inhibitors binding, there has been no research systematically evaluating the effects of alternative one-point mutations on the hydrolysis capacity of NDM-1. This study aims to identify which mutants could increase or decrease the effectiveness of antibiotics and ß-lactamase inhibitors toward bacteria. Firstly, 35 different variants with a high probability of emergence based on the PAM-1 matrix were constructed and then docked with 5 ligands, namely d-captopril, l-captopril, thiorphan, imipenem, and meropenem. The selected complexes underwent molecular dynamics simulation and free energy binding estimation, with the results showing that the substitutions at residues 122 and 124 most influenced the binding ability of NDM-1 toward inhibitors and antibiotics. The H122R mutant decreases the binding ability between d-captopril and NDM-1 and diminishes the effectiveness of this antibiotic toward Enterobacteriaceae. However, the H122R mutant has a contrary impact on thiorphan, which should be tested in vitro and in vivo in further experiments.

ThnL, a B12-dependent radical S-adenosylmethionine enzyme, catalyzes thioether bond formation in carbapenem biosynthesis.

Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B12-dependent radical S-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine. Neither of these functions has been observed previously in a B12-dependent radical SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond carbon-carbon bond formation or rearrangement. It is also the only radical SAM enzyme currently known to catalyze carbon-sulfur bond formation with only an rSAM Fe-S cluster and no additional auxiliary clusters.

The l,d-Transpeptidase LdtAb from Acinetobacter baumannii Is Poorly Inhibited by Carbapenems and Has a Unique Structural Architecture.

l,d-Transpeptidases (LDTs) are enzymes that catalyze reactions essential for biogenesis of the bacterial cell wall, including formation of 3-3 cross-linked peptidoglycan. Unlike the historically well-known bacterial transpeptidases, the penicillin-binding proteins (PBPs), LDTs are resistant to inhibition by the majority of ß-lactam antibiotics, with the exception of carbapenems and penems, allowing bacteria to survive in the presence of these drugs. Here we report characterization of LdtAb from the clinically important pathogen, Acinetobacter baumannii. We show that A. baumannii survives inactivation of LdtAb alone or in combination with PBP1b or PBP2, while simultaneous inactivation of LdtAb and PBP1a is lethal. Minimal inhibitory concentrations (MICs) of all 13 ß-lactam antibiotics tested decreased 2- to 8-fold for the LdtAb deletion mutant, while further decreases were seen for both double mutants, with the largest, synergistic effect observed for the LdtAb + PBP2 deletion mutant. Mass spectrometry experiments showed that LdtAb forms complexes in vitro only with carbapenems. However, the acylation rate of these antibiotics is very slow, with the reaction taking longer than four hours to complete. Our X-ray crystallographic studies revealed that LdtAb has a unique structural architecture and is the only known LDT to have two different peptidoglycan-binding domains.

QM/MM Simulations Reveal the Determinants of Carbapenemase Activity in Class A ß-Lactamases.

ß-lactam antibiotic resistance in Gram-negative bacteria, primarily caused by ß-lactamase enzymes that hydrolyze the ß-lactam ring, has become a serious clinical problem. Carbapenems were formerly considered "last resort" antibiotics because they escaped breakdown by most ß-lactamases, due to slow deacylation of the acyl-enzyme intermediate. However, an increasing number of Gram-negative bacteria now produce ß-lactamases with carbapenemase activity: these efficiently hydrolyze the carbapenem ß-lactam ring, severely limiting the treatment of some bacterial infections. Here, we use quantum mechanics/molecular mechanics (QM/MM) simulations of the deacylation reactions of acyl-enzyme complexes of eight ß-lactamases of class A (the most widely distributed ß-lactamase group) with the carbapenem meropenem to investigate differences between those inhibited by carbapenems (TEM-1, SHV-1, BlaC, and CTX-M-16) and those that hydrolyze them (SFC-1, KPC-2, NMC-A, and SME-1). QM/MM molecular dynamics simulations confirm the two enzyme groups to differ in the preferred acyl-enzyme orientation: carbapenem-inhibited enzymes favor hydrogen bonding of the carbapenem hydroxyethyl group to deacylating water (DW). QM/MM simulations of deacylation give activation free energies in good agreement with experimental hydrolysis rates, correctly distinguishing carbapenemases. For the carbapenem-inhibited enzymes, free energies for deacylation are significantly higher than for the carbapenemases, even when the hydroxyethyl group was restrained to prevent interaction with the DW. Analysis of these simulations, and additional simulations of mutant enzymes, shows how factors including the hydroxyethyl orientation, the active site volume, and architecture (conformations of Asn170 and Asn132; organization of the oxyanion hole; and the Cys69-Cys238 disulfide bond) collectively determine catalytic efficiency toward carbapenems.

Conformational flexibility in carbapenem hydrolysis drives substrate specificity of the class D carbapenemase OXA-24/40.

The evolution of multidrug resistance in Acinetobacter spp. increases the risk of our best antibiotics losing their efficacy. From a clinical perspective, the carbapenem-hydrolyzing class D ß-lactamase subfamily present in Acinetobacter spp. is particularly concerning because of its ability to confer resistance to carbapenems. The kinetic profiles of class D ß-lactamases exhibit variability in carbapenem hydrolysis, suggesting functional differences. To better understand the structure-function relationship between the carbapenem-hydrolyzing class D ß-lactamase OXA-24/40 found in Acinetobacter baumannii and carbapenem substrates, we analyzed steady-state kinetics with the carbapenem antibiotics meropenem and ertapenem and determined the structures of complexes of OXA-24/40 bound to imipenem, meropenem, doripenem, and ertapenem, as well as the expanded-spectrum cephalosporin cefotaxime, using X-ray crystallography. We show that OXA-24/40 exhibits a preference for ertapenem compared with meropenem, imipenem, and doripenem, with an increase in catalytic efficiency of up to fourfold. We suggest that superposition of the nine OXA-24/40 complexes will better inform future inhibitor design efforts by providing insight into the complicated and varying ways in which carbapenems are selected and bound by class D ß-lactamases.

Discovery of an Effective Small-Molecule Allosteric Inhibitor of New Delhi Metallo-ß-lactamase (NDM).

To identify novel inhibitors of the carbapenemase New Delhi metallo-ß-lactamase (NDM) as possible therapeutic compounds, we conducted a high-throughput screen of a 43,358-compound library. One of these compounds, a 2-quinazolinone linked through a diacylhydrazine to a phenyl ring (QDP-1) (IC50 = 7.9 ± 0.5 µM), was characterized as a slow-binding reversible inhibitor (Kiapp = 4 ± 2 µM) with a noncompetitive mode of inhibition in which substrate and inhibitor enhance each other's binding affinity. These studies, along with differential scanning fluorimetry, zinc quantitation, and selectivity studies, support an allosteric mechanism of inhibition. Cotreatment with QDP-1 effectively lowers minimum inhibitory concentrations of carbapenems for a panel of resistant Escherichia coli and Klebsiella pneumoniae clinical isolates expressing NDM-1 but not for those expressing only serine carbapenemases. QDP-1 represents a novel allosteric approach for NDM drug development for potential use alone or with other NDM inhibitors to counter carbapenem resistance in enterobacterales.

A hexasaccharide from capsular polysaccharide of carbapenem-resistant Klebsiella pneumoniae KN2 is a ligand of Toll-like receptor 4.

Klebsiella pneumoniae serotype KN2 is a carbapenem-resistant strain and leads to the health care-associated infections, such as bloodstream infections. Its capsular polysaccharide (CPS) was isolated and cleaved by a specific enzyme from a bacteriophage into a hexasaccharide-repeating unit. With GC-MS, NMR, and Mass analyses, the structure of KN2 CPS was determined to be {→3)-ß-D-Glcp-(1→3)-[α-D-GlcpA-(1→4)-ß-D-Glcp-(1→6)]-α-D-Galp-(1→6)-ß-D-Galp-(1→3)-ß-D-Galp-(1→}n. We demonstrated that 1 µg/mL CPS could stimulate J774A.1 murine macrophages to release tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in vitro. Also, we proved that KN2 CPS induced the immune response through Toll-like receptor 4 (TLR4) in the human embryonic kidney (HEK)-293 cells. Strikingly, the hexasaccharide alone shows the same immune response as the CPS, suggesting that the hexasaccharide can shape the adaptive immunity to be a potential vaccine adjuvant. The glucuronic acid (GlcA) on other polysaccharides can affect the immune response, but the GlcA-reduced KN2 CPS and hexasaccharide still maintain their immunomodulatory activities.

A novel tricyclic ß-lactam exhibiting potent antibacterial activities against carbapenem-resistant Enterobacterales: Synthesis and structure-activity-relationships.

A series of tricyclic ß-lactams were synthesized and evaluated for in vitro antibacterial activities against carbapenem-resistant Enterobacterales (CREs). Starting from a reported tricyclic ß-lactam that combined the cephalosporin skeleton having a γ-lactone ring with a carboxylic acid group, which was reported as a unique partial structure of Lactivicin, we identified the compound which shows potent antibacterial activities against all tested CREs by introducing sulfoxide. In addition, the sulfoxide-introduced tricyclic ß-lactam also shows a strong therapeutic efficacy in the neutropenic mouse lung infection model. These results indicate that the tricyclic ß-lactam skeleton will show sufficient therapeutic performance in clinical use and therefore can serve as a scaffold in the search for new antibacterial agents against CREs.

Identification of related impurities in an oral pharmaceutical formulation of tebipenem pivoxil using ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

RATIONALE: Tebipenem pivoxil (TBPM-PI) has been developed as the first oral carbapenem drug in the world to treat otolaryngological and respiratory infections in pediatric patients. Due to its structural properties and external factors, some related impurities, which may cause side effects in patients, might be formed during the synthesis and storage of TBPM-PI. It was vital to rapidly separate and identify the related impurities to guarantee the safe use of TBPM-PI. METHODS: A method using ultra-high-performance liquid chromatography (UHPLC) coupled with quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) was developed to separate and detect TBPM-PI and related impurities in an oral pharmaceutical formulation. LC/MS and MS/MS spectra of these compounds in the formulation were acquired to confirm their elemental compositions and propose their structures based on LC/MS data and fragmentation pathways of available reference substances. RESULTS: LC/MS parameters and MS/MS fragmentation pathways of reference substances of TBPM-PI and related impurities were summarized in detail. Based on this, a total of 23 related impurities were found and characterized in the oral pharmaceutical formulation. Eight of these were verified by comparison with reference substances and the structures of the other 15 were proposed for the first time. In addition, four of these compounds were produced by the reaction of excipients and pre-existing related impurities. CONCLUSIONS: A UHPLC/QTOF-MS method was established and used for the separation and identification of 23 related impurities in a TBPM-PI oral pharmaceutical formulation. Moreover, it was proved that new related impurities could be produced by the reaction of excipients in the pharmaceutical formulation and related impurities in the corresponding active pharmaceutical ingredient (API).

Evaluating the covalent binding of carbapenems on BlaC using noncovalent interactions.

Carbapenems, as irreversible covalent binders and slow substrates to the class A ß-lactamase (BlaC) of Mycobacterium tuberculosis, can inhibit BlaC to hydrolyze the ß-lactam drugs which are used to control tuberculosis. Their binding on BlaC involves covalent bonding and noncovalent interaction. We introduce a hypothesis that the noncovalent interactions dominate the difference of binding free energies for covalent ligands based on the assumption that their covalent bonding energies are the same. MM/GBSA binding free energies calculated from the noncovalent interactions provided a threshold with respect to the experimental kinetic data, to select slow carbapenem substrates which were either constructed using the structural units of experimentally identified carbapenems or obtained from the similarity search over the ZINC15 database. Combining molecular docking with consensus scoring and molecular dynamics simulation with MM/GBSA binding free energy calculations, a computational protocol was developed from which several new tight-binding carbapenems were theoretically identified.

Synthesis of Carbapenems Containing Peptidoglycan Mimetics and Inhibition of the Cross-Linking Activity of a Transpeptidase of l,d Specificity.

The carbapenem class of ß-lactams has been optimized against Gram-negative bacteria producing extended-spectrum ß-lactamases by introducing substituents at position C2. Carbapenems are currently investigated for the treatment of tuberculosis as these drugs are potent covalent inhibitors of l,d-transpeptidases involved in mycobacterial cell wall assembly. The optimization of carbapenems for inactivation of these unusual targets is sought herein by exploiting the nucleophilicity of the C8 hydroxyl group to introduce chemical diversity. As ß-lactams are structure analogs of peptidoglycan precursors, the substituents were chosen to increase similarity between the drug and the substrate. Fourteen peptido-carbapenems were efficiently synthesized. They were more effective than the reference drug, meropenem, owing to the positive impact of a phenethylthio substituent introduced at position C2 but the peptidomimetics added at position C8 did not further improve the activity. Thus, position C8 can be modified to modulate the pharmacokinetic properties of highly efficient carbapenems.

New Carbapenemase Inhibitors: Clearing the Way for the ß-Lactams.

Carbapenem resistance is a major global health problem that seriously compromises the treatment of infections caused by nosocomial pathogens. Resistance to carbapenems mainly occurs via the production of carbapenemases, such as VIM, IMP, NDM, KPC and OXA, among others. Preclinical and clinical trials are currently underway to test a new generation of promising inhibitors, together with the recently approved avibactam, relebactam and vaborbactam. This review summarizes the main, most promising carbapenemase inhibitors synthesized to date, as well as their spectrum of activity and current stage of development. We particularly focus on ß-lactam/ß-lactamase inhibitor combinations that could potentially be used to treat infections caused by carbapenemase-producer pathogens of critical priority. The emergence of these new combinations represents a step forward in the fight against antimicrobial resistance, especially in regard to metallo-ß-lactamases and carbapenem-hydrolysing class D ß-lactamases, not currently inhibited by any clinically approved inhibitor.

Transcriptome-based design of antisense inhibitors potentiates carbapenem efficacy in CRE Escherichia coli.

In recent years, the prevalence of carbapenem-resistant Enterobacteriaceae (CRE) has risen substantially, and the study of CRE resistance mechanisms has become increasingly important for antibiotic development. Although much research has focused on genomic resistance factors, relatively few studies have examined CRE pathogens through changes in gene expression. In this study, we examined the gene expression profile of a CRE Escherichia coli clinical isolate that is sensitive to meropenem but resistant to ertapenem to explore transcriptomic contributions to resistance and to identify gene knockdown targets for carbapenem potentiation. We sequenced total and short RNA to analyze the gene expression response to ertapenem or meropenem treatment and found significant expression changes in genes related to motility, maltodextrin metabolism, the formate hydrogenlyase complex, and the general stress response. To validate these findings, we used our laboratory's Facile Accelerated Specific Therapeutic (FAST) platform to create antisense peptide nucleic acids (PNAs), gene-specific molecules designed to inhibit protein translation. PNAs were designed to inhibit the pathways identified in our transcriptomic analysis, and each PNA was then tested in combination with each carbapenem to assess its effect on the antibiotics' minimum inhibitory concentrations. We observed significant PNA-antibiotic interaction with five different PNAs across six combinations. Inhibition of the genes hycA, dsrB, and bolA potentiated carbapenem efficacy in CRE E. coli, whereas inhibition of the genes flhC and ygaC conferred added resistance. Our results identify resistance factors and demonstrate that transcriptomic analysis is a potent tool for designing antibiotic PNA.

Amplification Curve Analysis: Data-Driven Multiplexing Using Real-Time Digital PCR.

Information about the kinetics of PCR reactions is encoded in the amplification curve. However, in digital PCR (dPCR), this information is typically neglected by collapsing each amplification curve into a binary output (positive/negative). Here, we demonstrate that the large volume of raw data obtained from real-time dPCR instruments can be exploited to perform data-driven multiplexing in a single fluorescent channel using machine learning methods, by virtue of the information in the amplification curve. This new approach, referred to as amplification curve analysis (ACA), was shown using an intercalating dye (EvaGreen), reducing the cost and complexity of the assay and enabling the use of melting curve analysis for validation. As a case study, we multiplexed 3 carbapenem-resistant genes to show the impact of this approach on global challenges such as antimicrobial resistance. In the presence of single targets, we report a classification accuracy of 99.1% (N = 16188), which represents a 19.7% increase compared to multiplexing based on the final fluorescent intensity. Considering all combinations of amplification events (including coamplifications), the accuracy was shown to be 92.9% (N = 10383). To support the analysis, we derived a formula to estimate the occurrence of coamplification in dPCR based on multivariate Poisson statistics and suggest reducing the digital occupancy in the case of multiple targets in the same digital panel. The ACA approach takes a step toward maximizing the capabilities of existing real-time dPCR instruments and chemistries, by extracting more information from data to enable data-driven multiplexing with high accuracy. Furthermore, we expect that combining this method with existing probe-based assays will increase multiplexing capabilities significantly. We envision that once emerging point-of-care technologies can reliably capture real-time data from isothermal chemistries, the ACA method will facilitate the implementation of dPCR outside of the lab.

Ruthenium complexes as prospective inhibitors of metallo-ß-lactamases to reverse carbapenem resistance.

The widespread prevalence of metallo-ß-lactamase (MßL)-mediated pathogens has seriously caused a loss of efficacy of carbapenem antibacterials, the last resort for the treatment of severe infectious diseases. The development of effective MßL inhibitors is an ideal alternative to restore the efficacy of carbapenems. Here we report that Ru complexes can irreversibly inhibit clinically relevant B1 subclass MßLs (NDM-1, IMP-1 and VIM-2) and potentiate meropenem efficacy against MßL-expressing bacteria in vitro and in a mice infection model. The Cys208 residue at the Zn(ii)-binding site and Met67 residue at the ß-hairpin loop of an enzyme active pocket are critical for Ru complexes to inhibit NDM-1, which was verified by enzyme kinetics, thermodynamics, NDM-1-C208A mutation and MALDI-TOF-MS analysis. This study will undoubtedly aid efforts to develop metal-based MßL inhibitors in combination with carbapenems to deal with the clinical crisis of carbapenem-resistant E. coli harboring MßLs.

The stability of carbapenems before and after admixture to PMMA-cement used for replacement surgery caused by Gram-negative bacteria.

BACKGROUND: Prosthetic joint infection (PJI) is a serious complication of orthopedic implant surgery. Treatment often includes the use of an antibiotic-loaded Polymethyl methacrylate (PMMA) bone cement spacer. Several antibiotics are commonly used for the preparation of these spacers, but due to the increasing number of infections with resistant Gram-negative bacteria, there is a need for the use of carbapenem antibiotics such as meropenem and imipenem as drugs of last resort. Unfortunately, the reaction heat generated during the preparation of the bone cement can be a major problem for the stability of these antibiotics. In the present study, the stability of meropenem and imipenem was tested before and after the admixture to PMMA bone cements. METHODS: High-performance liquid chromatography with ion-pairing reversed-phase separation and spectrophotometric detection was used for analysis. Stability tests with meropenem and imipenem were performed with antibiotics in solution and solid form at different temperatures (37 °C, 45 °C, 60 °C, 90 °C) and times (30 min, 60 min, 120 min). To test the stability of both antibiotics in PMMA after exposure to the reaction heat during polymerization, three different bone cements were used to generate specimens that contained defined amounts of antibiotics. Reaction heat was measured. The form bodies were mechanically crushed and aliquots were dissolved in ethyl acetate. Samples were prepared for HPLC DAD analysis. RESULTS: Meropenem and imipenem showed the highest degradation levels after heat stressed in solution, with maximum levels of 75% and 95%, respectively. In solid form, degradation levels decreased dramatically for meropenem (5%) and imipenem (13%). Stability tests of both carbapenems in bone cement showed that they remained largely stable during PMMA polymerization, with retrieved amounts of about 70% in Palacos® R and Copal® G+V, and between 80 and 90% in Copal® spacem. CONCLUSIONS: In contrast to the results of meropenem and imipenem in solution, both antibiotics remain stable in solid form and mostly stable in the cement after PMMA polymerization. The low degradation levels of both antibiotics after exposure to temperatures > 100 °C allow the conclusion that they can potentially be used for an application in PMMA cements.