The metallo-ß-lactamases strike back: emergence of taniborbactam escape variants.

Metallo-ß-lactamases (MBLs) have evolved relatively rapidly to become an international public health threat. There are no clinically available ß-lactamase inhibitors with activity against MBLs. This may change with the introduction of cefepime-taniborbactam. Herein, we review three manuscripts (S. I. Drusin, C. Le Terrier, L. Poirel, R. A. Bonomo, et al., Antimicrob Agents Chemother 68:e01168-23, 2024, https://doi.org/10.1128/aac.01168-23; C. Le Terrier, C. Viguier, P. Nordmann, A. J. Vila, and L. Poirel, Antimicrob Agents Chemother 68:e00991-23, 2024, https://doi.org/10.1128/aac.00991-23; D. Ono, M. F. Mojica, C. R. Bethel, Y. Ishii, et al., Antimicrob Agents Chemother 68:e01332-23, 2024, https://doi.org/10.1128/aac.01332-23) in which investigators describe elegant experiments to explore MBL/taniborbactam interactions and modifications to MBLs, in response, to reduce the affinity of taniborbactam. Challenges with MBL inhibition will not disappear; rather, they will evolve commensurate with advancements in medicinal chemistry.

Modification of carbapenemase inhibition test and comparison of its performance with NG-Test CARBA 5 for detection of carbapenemase-producing Enterobacterales.

AIMS: Adequately and accurately identifying carbapenemase-producing Enterobacterales (CPE) is vital for selecting appropriate antimicrobial therapy and implementing effective infection control measures. This study aims to optimize the phenotypic detection method of carbapenemase for routine diagnostics in clinical microbiology laboratories. METHODS AND RESULTS: Carbapenemase genes in 2665 non-duplicate CRE clinical strains collected from various regions of China were confirmed through whole-genome sequencing (WGS). The carbapenemase inhibition test (CIT) was conducted and interpreted using different methods and breakpoints, then compared with the NG-Test CARBA 5 for carbapenemase detection. The diagnostic performance of the CIT method was optimal when the carbapenemase types were determined by comparing the inhibition zone diameters of the imipenem disc with 3-aminophenylboronic acid (APB) plus ethylenediaminetetraacetic acid (EDTA) to those of the imipenem disc with either APB or EDTA alone, with a breakpoint of 4 mm. The overall sensitivities of the current CIT, the modified CIT, and NG-Test CARBA 5 were 91.4%, 94.9%, and 99.9%, respectively. For detecting isolates co-producing Klebsiella pneumoniae carbapenemase (KPC) and metallo-ß-lactamases (MBLs), the modified CIT method had higher sensitivity than the current method (70.0% vs. 53.3%), though this difference was not statistically significant (P = 0.063). The NG-Test CARBA 5 showed excellent performance for multi-carbapenemases diagnosis, with sensitivity and specificity of 97.1% and 100%, respectively. CONCLUSIONS: Optimizing and standardizing the CIT method for clinical use is necessary. It has certain advantages in diagnosing multi-carbapenemase and rare carbapenemase production. However, for identifying common carbapenemase types, the NG-Test CARBA 5 demonstrated superior performance.

Camel-Derived Nanobodies as Potent Inhibitors of New Delhi Metallo-ß-Lactamase-1 Enzyme.

The injudicious usage of antibiotics during infections caused by Gram-negative bacteria leads to the emergence of ß-lactamases. Among them, the NDM-1 enzyme poses a serious threat to human health. Developing new antibiotics or inhibiting ß-lactamases might become essential to reduce and prevent bacterial infections. Nanobodies (Nbs), the smallest antigen-binding single-domain fragments derived from Camelidae heavy-chain-only antibodies, targeting enzymes, are innovative alternatives to develop effective inhibitors. The biopanning of an immune VHH library after phage display has helped to retrieve recombinant antibody fragments with high inhibitory activity against recombinant-NDM-1 enzyme. Nb02NDM-1, Nb12NDM-1, and Nb17NDM-1 behaved as uncompetitive inhibitors against NDM-1 with Ki values in the nM range. Remarkably, IC50 values of 25.0 nM and 8.5 nM were noted for Nb02NDM-1 and Nb17NDM-1, respectively. The promising inhibition of NDM-1 by Nbs highlights their potential application in combating particular Gram-negative infections.

In vitro potency of xeruborbactam in combination with multiple ß-lactam antibiotics in comparison with other ß-lactam/ß-lactamase inhibitor (BLI) combinations against carbapenem-resistant and extended-spectrum ß-lactamase-producing Enterobacterales.

Recently, several ß-lactam (BL)/ß-lactamase inhibitor (BLI) combinations have entered clinical testing or have been marketed for use, but limited direct comparative studies of their in vitro activity exist. Xeruborbactam (XER, also known as QPX7728), which is undergoing clinical development, is a cyclic boronate BLI with potent inhibitory activity against serine (serine ß-lactamase) and metallo-ß-lactamases (MBLs). The objectives of this study were (i) to compare the potency and spectrum of ß-lactamase inhibition by various BLIs in biochemical assays using purified ß-lactamases and in microbiological assays using the panel of laboratory strains expressing diverse serine and metallo-ß-lactamases and (ii) to compare the in vitro potency of XER in combination with multiple ß-lactam antibiotics to that of other BL/BLI combinations in head-to-head testing against recent isolates of carbapenem-resistant Enterobacterales (CRE). Minimal inhibitory concentrations (MICs) of XER combinations were tested with XER at fixed 4 or 8 µg/mL, and MIC testing was conducted in a blinded fashion using Clinical and Laboratory Standards Institute reference methods. Xeruborbactam and taniborbactam (TAN) were the only BLIs that inhibited clinically important MBLs. The spectrum of activity of xeruborbactam included several MBLs identified in Enterobacterales, e.g., and various IMP enzymes and NDM-9 that were not inhibited by taniborbactam. Xeruborbactam potency against the majority of purified ß-lactamases was the highest in comparison with other BLIs. Meropenem-xeruborbactam (MEM-XER, fixed 8 µg/mL) was the most potent combination against MBL-negative CRE with MIC90 values of 0.125 µg/mL. MEM-XER and cefepime-taniborbactam (FEP-TAN) were the only BL/BLIs with activity against MBL-producing CREs; with MEM-XER (MIC90 of 1 µg/mL) being at least 16-fold more potent than FEP-TAN (MIC90 of 16 µg/mL). MEM-XER MIC values were ≤8 µg/mL for >90% of CRE, including both MBL-negative and MBL-positive isolates, with FEP-TAN MIC of >8 µg/mL. Xeruborbactam also significantly enhanced potency of other ß-lactam antibiotics, including cefepime, ceftolozane, ceftriaxone, aztreonam, piperacillin, and ertapenem, against clinical isolates of Enterobacterales that carried various class A, class C, and class D extended-spectrum ß-lactamases and carbapenem-resistant Enterobacterales, including metallo-ß-lactamase-producing isolates. These results strongly support further clinical development of xeruborbactam combinations.

Inhibitors for metallo-ß-lactamases from the B1 and B3 subgroups provide an avenue to combat a major mechanism of antibiotic resistance.

Metallo-ß-lactamases (MBLs) are a group of Zn(II)-dependent enzymes that pose a major threat to global health. They are linked to an increasing number of multi-drug resistant bacterial pathogens, but no clinically useful inhibitor is yet available. Since ß-lactam antibiotics, which are inactivated by MBLs, constitute ∼65% of all antibiotics used to treat infections, the search for clinically relevant MBL inhibitors is urgent. Here, derivatives of a 2-amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile (1a) were synthesised and their inhibitory effects assessed against prominent representatives of the MBL family. Several compounds are potent inhibitors of each MBL tested, making them promising candidates for the development of broad-spectrum drug leads. In particular, compound 5f is highly potent across the MBL family, with Ki values in the low µM range. Furthermore, this compound also appears to display synergy in combination with antibiotics such as penicillin G, cefuroxime or meropenem. This molecule thus represents a promising starting point to develop new drugs to inhibit a major mechanism of antibiotic resistance.

Molecular characteristics of clinical IMP-producing Klebsiella pneumoniae isolates: novel IMP-90 and integron In2147.

BACKGROUND: Since the first report of carbapenem-resistant Klebsiella pneumoniae isolates in China in 2007, the prevalence of CRKP and CRE has increased significantly. However, the molecular characteristics of IMP-producing Klebsiella pneumoniae (IMPKp) are rarely reported. METHODS: A total of 29 IMPKp isolates were collected from a Chinese tertiary hospital from 2011 to 2017. Clinical IMPKp were identified by VITEK®MS, and further analyzed by whole-genome DNA sequencing with HiSeq and PacBio RSII sequencer. Sequencing data were analyzed using CSI Phylogeny 1.4, Resfinder, PlasmidFinder and the MLST tool provided by the Centre for Genomic Epidemiology. The analysis results were visualized using iTOL editor v1_1. The open reading frames and pseudogenes were predicted using RAST 2.0 combined with BLASTP/BLASTN searches against the RefSeq database. The databases CARD, ResFinder, ISfinder, and INTEGRALL were performed for annotation of the resistance genes, mobile elements, and other features. The types of blaIMP in clinical isolates were determined by BIGSdb-Pasteur. Integrons were drawn by Snapgene, and the gene organization diagrams were drawn by Inkscape 0.48.1. RESULTS: Four novel ST type, including ST5422, ST5423, ST5426 and ST5427 were identified. The IMP-4 and IMP-1 were the dominant IMP type. The majority of blaIMP-carrying plasmids belonged to IncN and IncHI5. Two novel blaIMP-carrying integrons (In2146 and In2147) were uncovered. A novel variant blaIMP-90 presented in novel integron In2147 has been identified. CONCLUSIONS: IMPKp showed low prevalence in China. Novel molecular characteristics of IMPKp have been identified. Continuous monitoring of IMPKp shall also be carried out in the future.

Dihydroxyphenyl-substituted thiosemicarbazone: A potent scaffold for the development of metallo-ß-lactamases inhibitors and antimicrobial.

The superbug infection mediated by metallo-ß-lactamases (MßLs) has grown into anemergent health threat, and development of MßL inhibitors is an ideal strategy to combat the infection. In this work, twenty-five thiosemicarbazones 1a-e, 2a-e, 3a-e, 4a-d, 5a-d and 6a-b were synthesized and assayed against MßLs ImiS, NDM-1 and L1. The gained molecules specifically inhibited NDM-1 and ImiS, exhibiting an IC50 value in the range of 0.37-21.35 and 0.45-8.76 µM, and 2a was found to be the best inhibitor, with an IC50 of 0.37 and 0.45 µM, respectively, using meropenem (MER) as substrate. Enzyme kinetics and dialysis tests revealed and confirmed by ITC that 2a is a time-and dose-dependent inhibitor of ImiS and NDM-1, it competitively and reversibly inhibited ImiS with a Ki value of 0.29 µM, but irreversibly inhibited NDM-1. Structure-activity relationship disclosed that the substitute dihydroxylbenzene significantly enhanced inhibitory activity of thiosemicarbazones on ImiS and NDM-1. Most importantly, 1a-e, 2a-e and 3a-b alone more strongly sterilized E. coli-ImiS and E. coli-NDM-1 than the MER, displaying a MIC value in the range of 8-128 µg/mL, and 2a was found to be the best reagent with a MIC of 8 and 32 µg/mL. Also, 2a alone strongly sterilized the clinical isolates EC01, EC06-EC08, EC24 and K. pneumonia-KPC-NDM, showing a MIC value in the range of 16-128 µg/mL, and exhibited synergistic inhibition with MER on these bacteria tested, resulting in 8-32-fold reduction in MIC of MER. SEM images shown that the bacteria E. coli-ImiS, E. coli-NDM-1, EC24, K. pneumonia-KPC and K. pneumonia-KPC-NDM treated with 2a (64 µg/mL) suffered from distortion, emerging adhesion between individual cells and crumpled membranes. Mice tests shown that monotherapy of 2a evidently limited growth of EC24 cells, and in combination with MER, it significantly reduced the bacterial load in liver and spleen. Docking studies suggest that the 2,4-dihydroxylbenzene of 2a acts as zinc-binding group with the Zn(II) and the residual amino acids in CphA active center, tightly anchoring the inhibitor at active site. This work offered a promising scaffold for the development of MßLs inhibitors, specifically the antimicrobial for clinically drug-resistant isolates.

Aromatic Schiff bases confer inhibitory efficacy against New Delhi metallo-ß-lactamase-1 (NDM-1).

The irregular use of antibiotics has created a natural selection pressure for bacteria to adapt resistance. Bacterial resistance caused by metallo-ß-lactamases (MßLs) has been the most prevalent in terms of posing a threat to human health. The New Delhi metallo-ß-lactamase-1 (NDM-1) has been shown to be capable of hydrolyzing almost all ß-lactams. In this work, eight aromatic Schiff bases 1-8 were prepared and identified by enzyme kinetic assays to be the potent inhibitors of NDM-1 (except 4). These molecules exhibited a more than 95 % inhibition, and an IC50 value in the range of 0.13-19 µM on the target enzyme, and 3 was found to be the most effective inhibitor (IC50 = 130 nM). Analysis of structure-activity relationship revealed that the o-hydroxy phenyl improved the inhibitory activity of Schiff bases on NDM-1. The inhibition mode assays including isothermal titration calorimetry (ITC) disclosed that both compounds 3 and 5 exhibited a reversibly mixed inhibition on NDM-1, with a Ki value of 1.9 and 10.8 µM, respectively. Antibacterial activity tests indicated that a dose of 64 µg·mL-1 Schiff bases resulted in 2-128-fold reduction in MICs of cefazolin on E. coli producing NDM-1 (except 4). Cytotoxicity assays showed that both Schiff bases 3 and 5 have low cytotoxicity on the mouse fibroblast (L929) cells at a concentration of up to 400 µM. Docking studies suggested that the hydroxyl group interacts with Gln123 and Glu152 of NDM-1, and the amino groups interact with the backbone amide groups of Glu152 and Asp223. This study provided a novel scaffold for the development of NDM-1 inhibitors.

A Modified Vancomycin Molecule Confers Potent Inhibitory Efficacy against Resistant Bacteria Mediated by Metallo-ß-Lactamases.

Multidrug-resistant bacterial infections mediated by metallo-ß-lactamases (MßLs) have grown into an emergent health threat, and development of novel antimicrobials is an ideal strategy to combat the infections. Herein, a novel vancomycin derivative Vb was constructed by conjugation of triazolylthioacetamide and vancomycin molecules, characterized by reverse-phase high performance liquid chromatography (HPLC) and confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The biological assays revealed that Vb effectively inhibited S. aureus and methicillin-resistant S. aureus (MRSA), gradually increased the antimicrobial effect of ß-lactam antibiotics (cefazolin, meropenem and penicillin G) and exhibited a dose-dependent synergistic antibacterial effect against eight resistant strains tested, which was confirmed by the time-kill curves determination. Most importantly, Vb increased the antimicrobial effect of meropenem against the clinical isolates EC08 and EC10 and E. coli producing ImiS and CcrA, resulting in a 4- and 8-fold reduction in MIC values, respectively, at a dose up to 32 µg/mL. This work offers a promising scaffold for the development of MßLs inhibitors, specifically antimicrobials for clinically drug-resistant isolates.

Analysis of Antibacterial Action of Mammalian Host-Defense Cathelicidins and Induction of Resistance to Them in MßL-Producing Pseudomonas aeruginosa.

Recombinant analogs of a number of natural host-defense mammalian cathelicidins were obtained and predominant mechanism of their antibacterial action was studied. The ability of cathelicidins to suppress the growth of Pseudomonas aeruginosa producing metallo-ß-lactamases (MßL) was studied, and the possibility of appearance of cathelicidin-resistant bacteria was evaluated. Among peptides with different structures and mechanisms of action, only the strains resistant to ChMAP-28 were not obtained, which indicated minimum risk of the development of natural resistance to this cathelicidin. High antibacterial activity, wide spectrum of action, and the absence of cross-resistance effects allow considering ChMAP-28 peptide as a candidate to be developed further as a therapeutic agent against MßL-producing bacteria.

Cefiderocol as Rescue Therapy for Acinetobacter baumannii and Other Carbapenem-resistant Gram-negative Infections in Intensive Care Unit Patients.

Ten critically ill patients with either bacteremia or ventilator-associated pneumonia caused by carbapenem-resistant Acinetobacter baumannii, Stenotrophomonas maltophilia, or New Delhi metallo-ß-lactamase-producing Klebsiella pneumoniae received cefiderocol. All strains had minimum inhibitory concentration ≤2 µg/mL. Thirty-day clinical success and survival rates were 70% and 90%, respectively. Two patients had a microbiological failure. Future prospective studies are warranted.

Specific Protein-Membrane Interactions Promote Packaging of Metallo-ß-Lactamases into Outer Membrane Vesicles.

Outer membrane vesicles (OMVs) act as carriers of bacterial products such as plasmids and resistance determinants, including metallo-ß-lactamases. The lipidated, membrane-anchored metallo-ß-lactamase NDM-1 can be detected in Gram-negative OMVs. The soluble domain of NDM-1 also forms electrostatic interactions with the membrane. Here, we show that these interactions promote its packaging into OMVs produced by Escherichia coli. We report that favorable electrostatic protein-membrane interactions are also at work in the soluble enzyme IMP-1 while being absent in VIM-2. These interactions correlate with an enhanced incorporation of IMP-1 compared to VIM-2 into OMVs. Disruption of these interactions in NDM-1 and IMP-1 impairs their inclusion into vesicles, confirming their role in defining the protein cargo in OMVs. These results also indicate that packaging of metallo-ß-lactamases into vesicles in their active form is a common phenomenon that involves cargo selection based on specific molecular interactions.

Intermolecular interactions of the extended recognition site of VIM-2 metallo-ß-lactamase with 1,2,4-triazole-3-thione inhibitors. Validations of a polarizable molecular mechanics potential by ab initio QC.

Molecular dynamics on the complexes of inhibitors with Zn-metalloproteins are a privileged area of applications of polarizable molecular mechanics potentials. With which accuracy could these reproduce the QC intermolecular interaction energies in the two mono-zinc cores and in the dizinc core, toward full-fledged MD simulations on the entire protein complexes? We considered the complexes of the extended recognition site of a Zn-dependent metallo-ß-lactamase, VIM-2, produced by bacteria responsible for nosocomial infections, with five newly synthesized inhibitors sharing an original dizinc binding group, 1,2,4-triazole-3-thione (TZT). We considered the energy-minimized structures of each of the five VIM-2 complexes obtained with the SIBFA potential. Energy decomposition analyses (EDA) at the HF level enabled to compare the QC and the SIBFA ΔE values and their contributions in the zinc cores, with and without TZT, totaling 30 complexes. With one exception, the ΔE(QC) values were reproduced with relative errors <1.5%. We next considered the complex of the entire inhibitors with an extended model of VIM-2 recognition site, totaling up to 280 atoms. ΔE(SIBFA) could closely reproduce ΔE(QC). EDA analyses were resumed on the complexes of each inhibitor arm with its interacting VIM-2 residues. As a last step, EDA results at correlated levels were analyzed for the mono- and dizinc sites enabling comparisons with dispersion-augmented ΔE(SIBFA) and correlated multipoles and polarizabilities. Closely reproducing ΔE(QC) and the contrasting trends of its individual contributions should enable for dependable free energy perturbation studies and comparisons to recent experimental ΔG values, limiting as much as possible the reliance on error compensations.

Occurrence and Transmission of blaNDM-Carrying Enterobacteriaceae from Geese and the Surrounding Environment on a Commercial Goose Farm.

We investigated the prevalence and transmission of NDM-producing Enterobacteriaceae in fecal samples of geese and environmental samples from a goose farm in southern China. The samples were cultivated on MacConkey agar plates supplemented with meropenem. Individual colonies were examined for blaNDM, and blaNDM-positive bacteria were characterized based on whole-genome sequencing (WGS) data from the Illumina and Oxford Nanopore Technologies (ONT) platforms. Of 117 samples analyzed, the carriage rates for New Delhi metallo-ß-lactamase (NDM)-positive Enterobacteriaceae were 47.1, 18, and 50% in geese, inanimate environments (sewage, soil, fodder, and dust), and mouse samples, respectively. Two variants (blaNDM-1 and blaNDM-5, in 4 and 40 isolates, respectively) were found among 44 blaNDM-positive Enterobacteriaceae; these variants belonged to eight species, and Escherichia coli was the most prevalent (50%). WGS analysis revealed that blaNDM coexisted with diverse antibiotic resistance genes (ARGs). Population structure analysis showed that most E. coli and Enterobacter sp. isolates were highly heterogeneous, while most Citrobacter sp. and P. stuartii isolates possessed extremely high genetic similarities. In addition, blaNDM-5-positive ST4358/ST48 E. coli isolates were found to be clonally spread between geese and the environment and were highly genetically similar to those reported from ducks, farm environments, and humans in China. Plasmid analysis indicated that IncX3 pHNYX644-1-like (n = 40) and untypeable pM2-1-like plasmids (n = 4) mediated blaNDM spread. pM2-1-like plasmids possessed diverse ARGs, including blaNDM-1, the arsenical and mercury resistance operons, and the maltose operon. Our findings revealed that the goose farm is a reservoir for NDM-positive Enterobacteriaceae The blaNDM contamination of wild mice and the novel pM2-1-like plasmid described here likely adds to the risk for dissemination of blaNDM and associated resistance genes.IMPORTANCE Carbapenem-resistant bacteria, in particular NDM-producing Enterobacteriaceae, have become a great threat to global public. These bacteria have been found not only in hospital and community environments but also among food animal production chains, which are recognized as reservoirs for NDM-producing Enterobacteriaceae However, the dissemination of NDM-producing bacteria in waterfowl farms has been less well explored. Our study demonstrates that the horizontal spread of blaNDM-carrying plasmids and the partial clonal spread of blaNDM-positive Enterobacteriaceae contribute to the widespread contamination of blaNDM in the goose farm ecosystem, including mice. Furthermore, we found a novel and transferable blaNDM-1-carrying multidrug resistance (MDR) plasmid that possessed multiple environmental adaptation-related genes. The outcomes of this study contribute to a better understanding of the prevalence and transmission of blaNDM-carrying Enterobacteriaceae among diverse niches in the farm ecosystem.

Molecular study of metallo-ß-lactamases and integrons in Acinetobacter baumannii isolates from burn patients.

BACKGROUND: Productions of metallo-ß-lactamases enzymes are the most common mechanism of antibiotic resistance to all beta-lactam classes (except monobactams) in Acinetobacter baumannii. MBLs are usually associated with gene cassettes of integrons and spread easily among bacteria. The current study was performed to detect the genes encoding MBLs and integron structures in A. baumannii isolates from burn patients. METHODS: This study was performed on 106 non-duplicate A. baumannii isolates from burn patients referred to Shahid Motahari Hospital in Tehran. Antibiotic susceptibility of A. baumannii isolates was performed using disk diffusion and broth microdilution method in accordance with the CLSI guidelines. The presence of class 1 integron and associated gene cassettes as well as MBLs-encoding genes including blaVIM, and blaIMP were investigated using PCR and sequencing techniques. RESULTS: In this cross-sectional study all (100%) of the A. baumannii isolates examined were multidrug resistant. All isolates were sensitive to colistin and simultaneously all were resistant to imipenem. PCR assays showed the presence of blaVIM and blaIMP genes in 102 (96.2%) and 62 (58.5%) isolates of A. baumannii respectively. In addition, 62 (58.5%) of the A. baumannii isolates carried integron class 1, of which 49 (79.0%) were identified with at least one gene cassette. Three types of integron class 1 gene cassettes were identified including: arr2, cmlA5, qacE1 (2300 bp); arr-2, ereC, aadA1, cmlA7, qacE1 (4800 bp); and aac(3)-Ic, cmlA5 (2250 bp). CONCLUSION: A high prevalence of MBLs genes, especially blaVIM, was identified in the studied MDR A. baumannii isolates. In addition, most of the strains carried class 1 integrons. Furthermore, the gene cassettes arrays of integrons including cmlA5 and cmlA7 were detected, for the first time, in A. baumannii strains in Iran.

Dipyridyl-substituted thiosemicarbazone as a potent broad-spectrum inhibitor of metallo-ß-lactamases.

To combat the superbug infection caused by metallo-ß-lactamases (MßLs), a dipyridyl-substituted thiosemicarbazone (DpC), was identified to be the broad-spectrum inhibitor of MßLs (NDM-1, VIM-2, IMP-1, ImiS, L1), with an IC50 value in the range of 0.021-1.08 µM. It reversibly and competitively inhibited NDM-1 with a Ki value of 10.2 nM. DpC showed broad-spectrum antibacterial effect on clinical isolate K. pneumonia, CRE, VRE, CRPA and MRSA, with MIC value ranged from 16 to 32 µg/mL, and exhibited synergistic antibacterial effect with meropenem on MßLs-producing bacteria, resulting in a 2-16-, 2-8-, and 8-fold reduction in MIC of meropenem against EC-MßLs, EC01-EC24, K. pneumonia, respectively. Moreover, mice experiments showed that DpC also had synergistic antibacterial action with meropenem. In this work, DpC was identified to be a potent scaffold for the development of broad-spectrum inhibitors of MßLs.

Structure-guided optimization of D-captopril for discovery of potent NDM-1 inhibitors.

ß-lactam antibiotics have long been the mainstay for the treatment of bacterial infections. New Delhi metallo-ß-lactamase 1 (NDM-1) is able to hydrolyze nearly all ß-lactam antibiotics and even clinically used serine-ß-lactamase inhibitors. The wide and rapid spreading of NDM-1 gene among pathogenic bacteria has attracted extensive attention, therefore high potency NDM-1 inhibitors are urgently needed. Here we report a series of structure-guided design of D-captopril derivatives that can inhibit the activity of NDM-1 in vitro and at cellular levels. Structural comparison indicates the mechanisms of inhibition enhancement and provides insights for further inhibitor optimization.

Identification of metallo-ß-lactamases and AmpC production among Escherichia coli strains isolated from hemodialysis patients with urinary tract infection.

BACKGROUND: This study aimed to identify metallo-ß-lactamases (MBLs) and AmpC ß-lactamases-producing Escherichia coli isolates obtained from hemodialysis (HD) patients with urinary tract infections (UTI). METHODS AND RESULTS: A total of 257 HD patients with UTI were included in this study, from which 47 E. coli isolates were collected. Antibiotic susceptibility was tested by disc diffusion method. MBLs and AmpC production were phenotypically detected by imipenem-ethylenediaminetetracetate and cefoxitin/boronic acid assays, respectively. The presence of MBLs and AmpC genes was examined by polymerase chain reaction (PCR). Fosfomycin and ampicillin were the most and the least effective antibiotics against E. coli isolates, respectively. Moreover, 61.7% (29/47) of E. coli isolates were multidrug-resistant with seven different antibiotypes. Antibiotype V (AMP-CIP-IMP-MEM-CPD-CRO-CTX-GEN-LEV-SXT-TOB) was the most prevalent profile. Besides, 24 (51.1%) isolates were simultaneously resistant to imipenem and meropenem. Phenotypic assay showed MBL production in 16 (66.7%) of the 24 carbapenem-resistant E. coli isolates. The distribution of MBL genes in carbapenem-resistant E. coli was as follows: blaIMP 18 (72%), blaVIM 7 (28%), and blaNDM 1 (4%). AmpC was detected in 61.7% (29/47) of the isolates using the phenotypic method. The presence of AmpC genes was confirmed by PCR in only 26 of 29 (86.7%) AmpC producers. The frequencies of blaDHA-1, blaACC, and blaCMY-2 were 6 (20.7%), 11 (37.9%), and 21 (72.4%), respectively. CONCLUSIONS: The emergence of MBL and AmpC coproducing E. coli isolates calls for an urgent surveillance program for timely diagnosis and screening of these genes in our healthcare systems.

N-acylhydrazones confer inhibitory efficacy against New Delhi metallo-ß-lactamase-1.

The expression of ß-lactamases, especially metallo-ß-lactamases (MßLs) in bacteria is one of the main causes of drug resistance. In this work, an effective N-acylhydrazone scaffold as MßL inhibitor was constructed and characterized. The biological activity assays indicated that the synthesized N-acylhydrazones 1-11 preferentially inhibited MßL NDM-1, and 1 was found to be the most effective inhibitor with an IC50 of 1.2 µM. Analysis of IC50 data revealed a structure-activity relationship, which is that the pyridine and hydroxylbenzene substituents at 2-position improved inhibition of the compounds on NDM-1. ITC and enzyme kinetics assays suggested that it reversibly and competitively inhibited NDM-1 (Ki = 0.29 ± 0.05 µM). The synthesized N-acylhydrazones showed synergistic antibacterial activities with meropenem, reduced 4-16-fold MIC of meropenem on NDM-1- producing E. coli BL21 (DE3), while 1 restored 4-fold activity of meropenem on K. pneumonia expressing NDM-1 (NDM-K. pneumoniae). The mice experiments suggested that 1 combined meropenem to fight against NDM-K. pneumoniae infection in the spleen and liver. Cytotoxicity assays showed that 1 and 2 have low cytotoxicity. This study offered a new framework for the development of NDM-1 inhibitors.

Diaryl-substituted thiosemicarbazone: A potent scaffold for the development of New Delhi metallo-ß-lactamase-1 inhibitors.

The superbug infection caused by New Delhi metallo-ß-lactamase (NDM-1) has become an emerging public health threat. Inhibition of NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. A potent scaffold, diaryl-substituted thiosemicarbazone, was constructed and assayed with metallo-ß-lactamases (MßLs). The obtained twenty-six molecules specifically inhibited NDM-1 with IC50 0.038-34.7 µM range (except 1e, 2e, and 3d), and 1c is the most potent inhibitor (IC50 = 0.038 µM). The structure-activity relationship of synthetic thiosemicarbazones revealed that the diaryl-substitutes, specifically 2-pyridine and 2-hydroxylbenzene improved inhibitory activities of the inhibitors. The thiosemicarbazones exhibited synergistic antimycobacterial actions against E. coli-NDM-1, resulted a 2-512-fold reduction in MIC of meropenem, while 1c restored 16-256-, 16-, and 2-fold activity of the antibiotic on clinical isolates ECs, K. pneumonia and P. aeruginosa harboring NDM-1, respectively. Also, mice experiments showed that 1c had a synergistic antibacterial ability with meropenem, reduced the bacterial load clinical isolate EC08 in the spleen and liver. This work provided a highly promising scaffold for the development of NDM-1 inhibitors.