Subclass B1 Research Articles

Relative inhibitory activities of the broad-spectrum β-lactamase inhibitor xeruborbactam in comparison with taniborbactam against metallo-β-lactamases produced in Escherichia coli and Pseudomonas aeruginosa.

Xeruborbactam is a newly developed β-lactamase inhibitor designed for metallo-β-lactamases (MBLs). This study assessed the relative inhibitory properties of this novel inhibitor in comparison with another MBL inhibitor, namely taniborbactam (TAN), against a wide range of acquired MBL produced either in Escherichia coli or Pseudomonas aeruginosa. As observed with taniborbactam, the combination of xeruborbactam (XER) with β-lactams, namely, ceftazidime, cefepime and meropenem, led to significantly decreased MIC values for a wide range of B1-type MBL-producing E. coli, including most recombinant strains producing NDM, VIM, IMP, GIM-1, and DIM-1 enzymes. Noteworthily, while TAN-based combinations significantly reduced MIC values of β-lactams for MBL-producing P. aeruginosa recombinant strains, those with XER were much less effective. We showed that this latter feature was related to the MexAB-OprM efflux pump significantly impacting MIC values when testing XER-based combinations in P. aeruginosa. The relative inhibitory concentrations (IC50 values) were similar for XER and TAN against NDM and VIM enzymes. Noteworthily, XER was effective against NDM-9, NDM-30, VIM-83, and most of IMP enzymes, although those latter enzymes were considered resistant to TAN. However, no significant inhibition was observed with XER against IMP-10, SPM-1, and SIM-1 as well as the representative subclass B2 and B3 enzymes, PFM-1 and AIM-1. The determination of the constant inhibition (Ki) of XER revealed a much higher value against IMP-10 than against NDM-1, VIM-2, and IMP-1. Hence, IMP-10 that differs from IMP-1 by a single amino-acid substitution (Val67Phe) can, therefore, be considered resistant to XER.

Clonal dissemination of Acinetobacter radioresistens among Humboldt penguins (Spheniscus humboldti) inhabiting a barren northern Peruvian island.

Acinetobacter spp. are often isolated from natural sources, but knowledge about their presence in wild animals is fragmented and uncomplete. The present study aimed to characterize a series of Acinetobacter radioresistens isolated from Humboldt penguins (Spheniscus humboldti). Fifteen Humboldt penguins from an inhabited northern Peruvian island were sampled. Microorganisms were identified byMALDI-TOF MS. Antibiotic susceptibility to 12 antimicrobial agents was established, and clonal relationships were determined. A representative isolate was selected for whole genome sequencing (WGS). A. radioresistens were isolated from the feces of 12 (80%) Humboldt penguins, being susceptible to all the antimicrobial agents tested, except eight cefotaxime-intermediate isolates. AllA.radioresistens were clonally related. WGS showed that the isolate belonged to ST1972, the presence of two chromosomal encoded carbapenemases (blaOXA-23 and a putative subclass B3 metallo-β-lactamase), and a series of point mutations in antibiotic-resistance related chromosomal genes, which were considered as polymorphisms. In addition, a few virulence factors, including acapsule-encoding operon, superoxide dismutases, catalases, phospholipases and a siderophore receptor were identified. The present results suggest that A. radioresistens may be a common member of the gut microbiota of Humboldt penguins, but further studies in other geographical areas are needed to establish this finding.

Relative inhibitory activities of the broad-spectrum β-lactamase inhibitor taniborbactam against metallo-β-lactamases.

Taniborbactam (TAN) is a novel broad-spectrum β-lactamase inhibitor with significant activity against subclass B1 metallo-β-lactamases (MBLs). Here, we showed that TAN exhibited an overall excellent activity against B1 MBLs including most NDM- and VIM-like as well as SPM-1, GIM-1, and DIM-1 enzymes, but not against SIM-1. Noteworthy, VIM-1-like enzymes (particularly VIM-83) were less inhibited by TAN than VIM-2-like. Like NDM-9, NDM-30 (also differing from NDM-1 by a single amino acid substitution) was resistant to TAN.

Phenotypic and genomic characterization of a non-pathogenic Epilithonimonas ginsengisoli isolated from diseased farmed rainbow trout (Oncorhynchus mykiss) in Chile.

Flavobacterial infection associated with diseased fish is caused by multiple bacterial species within the family Flavobacteriaceae. In the present study, the Chilean isolate FP99, from the gills of a diseased, farmed rainbow trout (Oncorhynchus mykiss), was characterized using phenotypic and genomic analyses. Additionally assessed was pathogenic activity. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that isolate FP99 belonged to the genus Epilithonimonas, an average nucleotide identity value of 100% was detected with the Chilean isolate identified as Epilithonimonas sp. FP211-J200. In silico genome analysis, mechanisms for toxins production, and superantigens, adhesion, or other genes associated with virulence were not detected. However, genes encoding proteins for antibiotic resistance were found, including the chrA gene and the nucleotide sequence that encodes for multiple antibiotic resistance MarC proteins. Furthermore, the blaESP-1 gene (87.85% aminoacidic sequence identity), encoding an extended-spectrum subclass B3 metallo-β-lactamase and conferring carbapenem-hydrolysing activity, and the tet(X) gene, which encodes a monooxygenase that catalyses the degradation of tetracycline-class antimicrobials were carried by this isolate. Phenotyping analyses also supported assignment as E. ginsengisoli. Challenge trials against healthy rainbow trout resulted in no observed pathogenic effect. Our findings identify for the first time the species E. ginsengisoli as associated with fish farming, suggesting that this isolate may be a component of the microbiota of the freshwater system. Notwithstanding, poor environmental conditions and any stressors associated with aquaculture situations or lesions caused by other pathogenic bacteria, such as F. psychrophilum, could favour the entry of E. ginsengisoli into rainbow trout.

Diversity of blaPOM in carbapenem-resistant opportunistic pathogenic Pseudomonas otitidis in municipal wastewater.

Metallo-β-lactamases (MBLs) encoding carbapenem resistance in wastewater are a well-known serious threat to human health. Twelve Pseudomonas otitidis isolates obtained from a municipal wastewater treatment plant (WWTP) in Hawaii were found to possess a subclass B3 MBL - POM (P. otitidis MBL), with a minimum inhibition concentration (MIC) range of 8-16 mg/L. The unrooted neighbor-joining phylogenetic tree showed that these blaPOM genes isolated in wastewater samples (n = 12) were distinctly different from other reference genes isolated from clinical, freshwater, animal, and soil samples except for isolates MR7, MR8, and MR11. MR7, MR8, and MR11 were found to have 4, 3, and 3 amino acid substitutions when compared to the type strain MC10330T and were closely clustered to the clinical reference genes. The meropenem hydrolysis experiment showed that isolates with multiple amino acid substitutions completely hydrolyzed 64 mg/L of meropenem in 7 h. The emergence of the opportunistic pathogen P. otitidis chromosomally encoding blaPOM in the treated municipal wastewater is an alarming call for the spread of this MBL in the environment. Further studies are required to understand the mechanism and regulation of this carbapenem-resistant β-lactamase in order to fill in the knowledge gap.

The in vitro inhibitory activity of polypyridine ligands towards subclass B1 metallo-β-lactamases

Antimicrobial resistance leads to the ineffectiveness of antimicrobials, hampering the ability to cure infections. Zinc-dependent metallo ß-lactamases (MBLs) producing bacteria represent a threat to public health due to limited therapeutic options. Zinc chelators are able to inhibit MBLs and have the potential to restore carbapenem susceptibility. Recently, polypyridine ligands named Bispicen (N,N’-Bis(2-pyridymethyl)-ethylenediamine), Trispicen (N,N,N’-Tris(2-pyridylmethyl)-ethylenediamine), TrispicenA (N,N,N’-tris([2-pyridylmethyl)-ethylenediamine-N’-acetic acid) and TrispicenDALA (N,N,N’-tris([2-pyridylmethyl)-ethylenediamine-N’-acetyl-D-alanyl-D-alanyl-D-alanine methyl ester) were evaluated for their ability to interfere with the Zn-ion network by interactions in the binding site of MBLs. We report on the inhibitory activity of these ligands towards three subclass B1 MBLs (NDM-1, VIM-1 and IMP-1) by kinetic analysis, demonstrating that all compounds, except TrispicenDALA, acted as competitive inhibitors towards NDM-1 and VIM-1. Bispicen was more active against VIM-1 with Ki value of 0.13 μM while NDM-1 was highly inhibited by Trispicen with Ki value of 0.08 μM. The IMP-1 enzyme was resistant to these compounds showing Ki and IC50 values higher than 500 μM. Docking study reveals that all compounds interact with residues of the MBL active site mainly by the formation of pi-interactions and chelation of both Zn ions. Polypyridine ligands deserve attention for their inhibitory potential towards NDM-1 and VIM-1.

Comprehensive screening of marine metabolites against class B1 metallo-β-lactamases of Klebsiella pneumoniae using two-pronged in silico approach

The emergence of antibiotic resistance is one of the major global threats in healthcare. Metallo-β-Lactamases (MBL) are a class of enzymes in bacteria that cleave β-lactam antibiotics and confer resistance. MBLs are further divided into subclasses B1, B2 and B3. Of these, subclasses B1-MBLs (including NDM-1, VIM-2 and IMP-1) constitute the clinically prevalent lactamases conferring resistance. To date, no effective drugs are available clinically against MBLs. In this work, we aim to identify potent inhibitors for the B1 subclass of MBL from available marine metabolites in Comprehensive Marine Natural Product database through integrated in silico approaches. We have used two methods, namely, the high-throughput strategy and the pharmacophore-based strategy to identify potential inhibitors from marine metabolites. High-throughput virtual screening identified N-methyl mycosporine-Ser, which had the highest binding affinity to NDM-1. The pharmacophore-based approach based on co-crystallized ligands identified makaluvic acid and didymellamide with higher binding affinity across B1-MBLs. Taking into account of the advantage of a pharmacophore model-based approach with higher binding affinity, we conclude that both makaluvic acid and didymellamide show potential broad-spectrum effects by binding to all three B1-MBL receptors. The study also indicates the need to take multiple in silico approaches to screen and identify novel inhibitors. Together, our study reveals promising inhibitors that can be identified from marine systems. Communicated by Ramaswamy H. Sarma

Design, Synthesis, and Biological Evaluation of New 1H-Imidazole-2-Carboxylic Acid Derivatives as Metallo-β-Lactamase Inhibitors

As one of important mechanisms to β-lactam antimicrobial resistance, metallo-β-lactamases (MBLs) have been receiving increasing worldwide attentions. Ambler subclass B1 MBLs are most clinically relevant, because they can hydrolyze almost all β-lactams with the exception of monobactams. However, it is still lacking of clinically useful drugs to combat MBL-medicated resistance. We previously identified 1H-imidazole-2-carboxylic acid as a core metal-binding pharmacophore (MBP) to target multiple B1 MBLs. Herein, we report structural optimization of 1H-imidazole-2-carboxylic acid and substituents. Structure-activity relationship (SAR) analyses revealed that replacement of 1H-imidazole-2-carboxylic acid with other structurally highly similar MBPs excepting thiazole-4-carboxylic acid resulted in decreased MBL inhibition. Further SAR studies identified more potent inhibitors to MBLs, of which 28 manifested IC50 values of 0.018 µM for both VIM-2 and VIM-5. The microbiological tests demonstrated that the most tested compounds showed improved synergistic effects; some compounds at 1 µg/ml were able to reduce meropenem MIC by at least 16-fold, which will be worth further development of new potent inhibitors particularly targeting VIM-type MBLs.

Biochemical Characterization of the Subclass B3 Metallo-β-Lactamase PJM-1 from Pseudoxanthomonas japonensis.

Biochemical properties of the novel subclass B3 metallo-β-lactamase (MBL) PJM-1 expressed in Pseudoxanthomonas japonensis, which is often isolated from the environment, were determined. The 906-bp blaPJM-1 gene in P. japonensis is a species-specific MBL gene, and PJM, with 301 predicted amino acids, has 81.8% amino acid identity with AIM-1. In this study, PJM-1 was recombinantly expressed and purified. PJM-1 showed a low catalytic activity against ceftazidime and cefepime, and it was strongly inhibited by EDTA.

Detection by metagenomic functional analysis and improvement by experimental evolution of β-lactams resistance genes present in oil contaminated soils

The spread of antibiotic resistance genes has become a global health concern identified by the World Health Organization as one of the greatest threats to health. Many of antimicrobial resistance determinants found in bacterial pathogens originate from environmental bacteria, so identifying the genes that confer resistance to antibiotics in different habitats is mandatory to better understand resistance mechanisms. Soil is one of the most diverse environments considered reservoir of antimicrobial resistance genes. The aim of this work is to study the presence of genes that provide resistance to antibiotics used in clinical settings in two oil contaminated soils by metagenomic functional analysis. Using fosmid vectors that efficiently transcribe metagenomic DNA, we have selected 12 fosmids coding for two class A β-lactamases, two subclass B1 and two subclass B3 metallo-β-lactamases, one class D β-lactamase and three efflux pumps that confer resistance to cefexime, ceftriaxone, meropenem and/or imipenem. In some of them, detection of the resistance required heterologous expression from the fosmid promoter. Although initially, these environmental genes only provide resistance to low concentrations of antibiotics, we have obtained, by experimental evolution, fosmid derivatives containing β-lactamase ORFs with a single base substitution, which substantially increase their β-lactamase activity and resistance level. None of the mutations affect β-lactamase coding sequences and are all located upstream of them. These results demonstrate the presence of enzymes that confer resistance to relevant β-lactams in these soils and their capacity to rapidly adapt to provide higher resistance levels.

Studies on the Reactions of Biapenem with VIM Metallo β-Lactamases and the Serine β-Lactamase KPC-2.

Carbapenems are important antibacterials and are both substrates and inhibitors of some β-lactamases. We report studies on the reaction of the unusual carbapenem biapenem, with the subclass B1 metallo-β-lactamases VIM-1 and VIM-2 and the class A serine-β-lactamase KPC-2. X-ray diffraction studies with VIM-2 crystals treated with biapenem reveal the opening of the β-lactam ring to form a mixture of the (2S)-imine and enamine complexed at the active site. NMR studies on the reactions of biapenem with VIM-1, VIM-2, and KPC-2 reveal the formation of hydrolysed enamine and (2R)- and (2S)-imine products. The combined results support the proposal that SBL/MBL-mediated carbapenem hydrolysis results in a mixture of tautomerizing enamine and (2R)- and (2S)-imine products, with the thermodynamically favoured (2S)-imine being the major observed species over a relatively long-time scale. The results suggest that prolonging the lifetimes of β-lactamase carbapenem complexes by optimising tautomerisation of the nascently formed enamine to the (2R)-imine and likely more stable (2S)-imine tautomer is of interest in developing improved carbapenems.

Characterization of GMB-1, a novel metallo-β-lactamase (MBL) found in three different Enterobacterales species.

To identify novel carbapenem resistance mechanisms and their potential to spread among clinical isolates. Four clinical isolates of Citrobacter freundii, Serratia marcescens and Raoultella planticola (n = 2) from one hospital in Central Germany were sent to the German National Reference Centre for Multidrug-resistant Gram-negative Bacteria for carbapenemase detection. Phenotypic tests indicated the presence of a metallo-β-lactamase (MBL), but PCR for various MBL genes could not identify any. Using WGS data, a putative bla gene was identified. Its carbapenemase activity was verified by heterologous expression in an Escherichia coli cloning strain, with subsequent MIC determination by broth microdilution, as well as by in vitro hydrolysis assays using purified enzyme. WGS indicated the presence of a putative β-lactamase with 48% amino acid identity to the subclass B1 MBL SPM-1. MIC studies confirmed that the novel enzyme formed a functional MBL, which was therefore designated as GMB-1 (German MBL). In vitro hydrolysis assays showed a lack of activity not only against aztreonam but also against ertapenem. WGS revealed that in all three species the blaGMB-1 gene was located on the chromosome as part of a genetic island with multiple ISs. The finding of GMB-1 once again shows that novel carbapenemases continue to emerge and make their way into clinically relevant species. The occurrence of GMB-1 in three different species demonstrates the extraordinary mobility of such genetic islands and their potential to spread carbapenemase genes into diverse genetic environments.

BioF is a novel B2 metallo-β-lactamase from Pseudomonas sp. isolated from an on-farm biopurification system.

Antimicrobial resistance represents a major global health concern and environmental bacteria are considered a source of resistance genes. Carbapenems are often used as the last antibiotic option to treat multidrug-resistant bacteria. Metallo-β-lactamases (MBLs) are able to render resistance to almost all β-lactam antibiotics, including carbapenems. Unfortunately, there are no inhibitors against MBLs for clinical use. Subclass B2 MBLs are the only enzymes working as strict carbapenemases, under-represented, encoded in chromosome genes and only functional as mono-zinc enzymes. Despite current efforts in MBLs inhibitor development, B2 carbapenemase activity is especially difficult to suppress, even in vitro. In this study we characterized BioF, a novel subclass B2 MBL identified in a new environmental Pseudomonas sp. strain isolated from an on-farm biopurification system (BPS). Although blaBioF is most likely a chromosomal gene, it is found in a genomic island and may represent a step previous to the horizontal transmission of B2 genes. The new B2 MBL is active as a mono-zinc enzyme and is a potent carbapenemase with incipient activity against some cephalosporins. BioF activity is not affected by excess zinc and is only inhibited at high metal chelator concentrations. The discovery and characterization of B2 MBL BioF as a potent carbapenemase in a BPS bacterial isolate emphasizes the importance of exploring antibiotic resistances existing in the environmental microbiota under the influence of human activities before they could emerge clinically.

Characterization of the novel plasmid-encoded MBL gene blaAFM-1, integrated into a blaIMP-45-bearing transposon Tn6485e in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate.

To characterize the novel subclass B1 MBL AFM-1, encoded by a blaIMP-45-bearing megaplasmid from a carbapenem-resistant Pseudomonas aeruginosa (CRPA) clinical isolate. CRPA HS17-127 and its transconjugant were discovered to carry blaAFM-1 in our previous study. blaAFM-1 and blaNDM-1 were cloned and expressed in Escherichia coli TOP10 and P. aeruginosa PAO1, respectively, to test the resistance phenotype. Kinetic studies were performed to elucidate the biochemical characteristics of the AFM-1 enzyme. Comparative genomic analysis was applied to investigate the genetic context of blaAFM-1. PAO1 transconjugant TcHS17-127 exhibited carbapenem resistance with an imipenem MIC of 64 mg/L. E. coli transformants with cloned blaAFM-1 or blaNDM-1 had increased MICs of all β-lactams tested (except aztreonam) and imipenem MICs of 4-8 mg/L. Kinetic studies showed that AFM-1 had greater catalytic efficiency against cephalosporins than carbapenems. blaAFM-1 was located on a 486 963 bp IncP-2 plasmid, pHS17-127, containing a 57.3 kb MDR Tn1403-derivative transposon, Tn6485e, which is genetically closest to the blaIMP-45-bearing Tn6485 transposon but has acquired an extra ISCR27n3-blaAFM-1 module. Multicentre surveillance of 605 P. aeruginosa clinical isolates identified three blaAFM carriers from different STs. Two of them co-carried blaAFM-1 and blaIMP-45. A BLAST search against the NCBI database showed six blaAFM carriers on various plasmids and the chromosomes of different Gram-negative species. The blaAFM-1 gene confers carbapenem resistance and has been captured in distinct species of non-fermenters. Co-carriage of blaAFM-1 and blaIMP-45 in an MDR transposon on a conjugative plasmid can be expected to promote further dissemination of blaMBLs.

2-Mercaptomethyl Thiazolidines (MMTZs) Inhibit All Metallo-β-Lactamase Classes by Maintaining a Conserved Binding Mode.

Metallo-β-lactamase (MBL) production in Gram-negative bacteria is an important contributor to β-lactam antibiotic resistance. Combining β-lactams with β-lactamase inhibitors (BLIs) is a validated route to overcoming resistance, but MBL inhibitors are not available in the clinic. On the basis of zinc utilization and sequence, MBLs are divided into three subclasses, B1, B2, and B3, whose differing active-site architectures hinder development of BLIs capable of "cross-class" MBL inhibition. We previously described 2-mercaptomethyl thiazolidines (MMTZs) as B1 MBL inhibitors (e.g., NDM-1) and here show that inhibition extends to the clinically relevant B2 (Sfh-I) and B3 (L1) enzymes. MMTZs inhibit purified MBLs in vitro (e.g., Sfh-I, Ki 0.16 μM) and potentiate β-lactam activity against producer strains. X-ray crystallography reveals that inhibition involves direct interaction of the MMTZ thiol with the mono- or dizinc centers of Sfh-I/L1, respectively. This is further enhanced by sulfur-π interactions with a conserved active site tryptophan. Computational studies reveal that the stereochemistry at chiral centers is critical, showing less potent MMTZ stereoisomers (up to 800-fold) as unable to replicate sulfur-π interactions in Sfh-I, largely through steric constraints in a compact active site. Furthermore, in silico replacement of the thiazolidine sulfur with oxygen (forming an oxazolidine) resulted in less favorable aromatic interactions with B2 MBLs, though the effect is less than that previously observed for the subclass B1 enzyme NDM-1. In the B3 enzyme L1, these effects are offset by additional MMTZ interactions with the protein main chain. MMTZs can therefore inhibit all MBL classes by maintaining conserved binding modes through different routes.

Detection of clinically important β-lactamases by using PCR.

Increasing antimicrobial resistance of nosocomial pathogens is becoming a serious threat to public health. To control the spread of this resistance, it is necessary to detect β-lactamase-producing organisms in the clinical setting. The aims of the study were to design a PCR assay for rapid detection of clinically encountered β-lactamase genes described in Enterobacteriaceae and Gram-negative non-fermenting bacteria. The functionality of proposed primers was verified using eight reference strains and 17 strains from our collection, which contained 29 different β-lactamase genes. PCR products of the test strains were confirmed by Sanger sequencing. Sequence analysis was performed using bioinformatics software Geneious. Overall, 67 pairs of primers for detecting 12 members of the class C β-lactamase family, 15 members of class A β-lactamases, six gene families of subclass B1, one member each of subclasses B2, B3 and class D β-lactamases were designed, of which 43 pairs were experimentally tested in vitro. All 29 β-lactamase genes, including 10 oxacillinase subgroups, were correctly identified by PCR. The proposed set of primers should be able to specifically detect 99.7% of analyzed β-lactamase subtypes and more than 79.8% of all described β-lactamase genes.

Molecular docking studies, in-silico ADMET predictions and synthesis of novel PEGA-nucleosides as antimicrobial agents targeting class B1 metallo-β-lactamases.

Class B1 metallo-β-lactamases (MBLs) are metalloenzymes found in drug resistant bacteria. The enzyme requires zinc ions, along with conserved amino acid coordination for nucleophilic attack of the lactam ring to induce hydrolysis and inactivation of β-lactam and some carbapenem antibiotics. To this date there are no clinically relevant class B1 MBL inhibitors, however L-captopril has shown significant results against NDM-1, the most difficult MBL to inhibit. Herein, we report the synthesis and evaluation of novel nucleoside analogues modified with polyethylene glycolamino (PEGA) as potential inhibitors for class B1 MBLs. Molecular dynamics simulations, using internal coordinate mechanics (ICM) algorithm, were performed on subclass B1 enzyme complex models screened with twenty-one possible PEGA-nucleosides. Analogue A, 3'-deoxy-3'-(2-(2-hydroxyethoxy)ethanamino)-β-D-xylofuranosyluracil showed superior binding, with high specificity to the conserved zinc ions in the class B1 MBL active site by utilizing key β-lactam mimic points in the uridine nucleobase. The PEGA moiety showed chelating activity with zinc and disrupted the metal-binding amino acid geometry. In all subclass B1 proteins tested, analogue A had the most effective inhibition when compared to penicillin or L-captopril. Chemical synthesis was performed by condensation of the corresponding keto ribonucleoside with PEGA, followed by enantioselective reduction of the formed imine to produce the amino derivative with desired configuration. Pharmacokinetic and pharmacodynamic screenings revealed that PEGA-pyrimidine nucleosides are not toxic, nor violate Lipinski's rules. These results suggested that analogue A can be proposed as a potential metalloenzyme inhibitor against the widespread antibiotic resistant bacteria and is worth further in vitro and in vivo investigations.

Complete whole-genome sequence of the novel Pseudomonas species strain TUM18999, isolated from a patient with a burn wound in Japan

ObjectivesPseudomonas is a Gram-negative bacterial genus with numerous member species. In this study, using whole-genome sequencing, we characterized a novel Pseudomonas sp. strain TUM18999, isolated as a pathogen from a human patient. MethodsThe TUM18999 strain was isolated from a patient’s burn wound. Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method. The whole-genome sequence was obtained using Miseq and MinION, and we conducted phylogenetic analysis based on single nucleotide polymorphisms of the core genome. ResultsAntimicrobial susceptibility testing revealed a high ceftazidime MIC (32 mg/L). Moreover, carbapenemase production was confirmed using the modified carbapenem inactivation method. We found that the complete genome of TUM18999 was 6,826,062 bp long, with 6175 coding sequences (CDS) and a DNA G+C content (non-plasmid) of 66.4 mol%. Consistent with the high similarities with the 16S rRNA sequences of P. otitidis MCC10330 (98.6%) and P. alcaligenes NBRC 14159 (99.2%), similarities (<90%) were also observed with the gyrB genes of both strains. The average nucleotide identities for P. alcaligenes NBRC 14159 and P. otitidis MCC10330 were also <90%. The core-genome single nucleotide polymorphism phylogenetic tree indicated that the TUM18999 strain was most closely related to P. otitidis MCC10330. In addition, the TUM18999 strain carried the novel gene, species-specific subclass B3 metallo-β-lactamase (MBL), and its similarities with P. alcaligenes metallo-β-lactamase-1 (PAM-1) and P. otitidis metallo-β-lactamase-1 (POM-1) were 90.24% and 73.14%, respectively. ConclusionWe characterized the complete whole genome sequence of the novel Pseudomonas sp. TUM18999 carrying the novel gene species-specific subclass B3 MBL.

Faropenem reacts with serine and metallo-β-lactamases to give multiple products.

Penems have demonstrated potential as antibacterials and β-lactamase inhibitors; however, their clinical use has been limited, especially in comparison with the structurally related carbapenems. Faropenem is an orally active antibiotic with a C-2 tetrahydrofuran (THF) ring, which is resistant to hydrolysis by some β-lactamases. We report studies on the reactions of faropenem with carbapenem-hydrolysing β-lactamases, focusing on the class A serine β-lactamase KPC-2 and the metallo β-lactamases (MBLs) VIM-2 (a subclass B1 MBL) and L1 (a B3 MBL). Kinetic studies show that faropenem is a substrate for all three β-lactamases, though it is less efficiently hydrolysed by KPC-2. Crystallographic analyses on faropenem-derived complexes reveal opening of the β-lactam ring with formation of an imine with KPC-2, VIM-2, and L1. In the cases of the KPC-2 and VIM-2 structures, the THF ring is opened to give an alkene, but with L1 the THF ring remains intact. Solution state studies, employing NMR, were performed on L1, KPC-2, VIM-2, VIM-1, NDM-1, OXA-23, OXA-10, and OXA-48. The solution results reveal, in all cases, formation of imine products in which the THF ring is opened; formation of a THF ring-closed imine product was only observed with VIM-1 and VIM-2. An enamine product with a closed THF ring was also observed in all cases, at varying levels. Combined with previous reports, the results exemplify the potential for different outcomes in the reactions of penems with MBLs and SBLs and imply further structure-activity relationship studies are worthwhile to optimise the interactions of penems with β-lactamases. They also exemplify how crystal structures of β-lactamase substrate/inhibitor complexes do not always reflect reaction outcomes in solution.

2-Mercaptomethyl-thiazolidines use conserved aromatic-S interactions to achieve broad-range inhibition of metallo-β-lactamases.

Infections caused by multidrug resistant (MDR) bacteria are a major public health threat. Carbapenems are among the most potent antimicrobial agents that are commercially available to treat MDR bacteria. Bacterial production of carbapenem-hydrolysing metallo-β-lactamases (MBLs) challenges their safety and efficacy, with subclass B1 MBLs hydrolysing almost all β-lactam antibiotics. MBL inhibitors would fulfil an urgent clinical need by prolonging the lifetime of these life-saving drugs. Here we report the synthesis and activity of a series of 2-mercaptomethyl-thiazolidines (MMTZs), designed to replicate MBL interactions with reaction intermediates or hydrolysis products. MMTZs are potent competitive inhibitors of B1 MBLs in vitro (e.g., Ki = 0.44 μM vs. NDM-1). Crystal structures of MMTZ complexes reveal similar binding patterns to the most clinically important B1 MBLs (NDM-1, VIM-2 and IMP-1), contrasting with previously studied thiol-based MBL inhibitors, such as bisthiazolidines (BTZs) or captopril stereoisomers, which exhibit lower, more variable potencies and multiple binding modes. MMTZ binding involves thiol coordination to the Zn(ii) site and extensive hydrophobic interactions, burying the inhibitor more deeply within the active site than d/l-captopril. Unexpectedly, MMTZ binding features a thioether–π interaction with a conserved active-site aromatic residue, consistent with their equipotent inhibition and similar binding to multiple MBLs. MMTZs penetrate multiple Enterobacterales, inhibit NDM-1 in situ, and restore carbapenem potency against clinical isolates expressing B1 MBLs. Based on their inhibitory profile and lack of eukaryotic cell toxicity, MMTZs represent a promising scaffold for MBL inhibitor development. These results also suggest sulphur–π interactions can be exploited for general ligand design in medicinal chemistry.