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Mode of Action of the Monobactam LYS228 and Mechanisms Decreasing In Vitro Susceptibility in Escherichia coli and Klebsiella pneumoniae.

ABSTRACT: The monobactam scaffold is attractive for the development of new agents to treat infections caused by drug-resistant Gram-negative bacteria because it is stable to metallo-?-lactamases (MBLs). However, the clinically used monobactam aztreonam lacks stability to serine ?-lactamases (SBLs) that are often coexpressed with MBLs. LYS228 is stable to MBLs and most SBLs. LYS228 bound purified Escherichia coli penicillin binding protein 3 (PBP3) similarly to aztreonam (derived acylation rate/equilibrium dissociation constant [k2/Kd ] of 367,504 s-1 M-1 and 409,229 s-1 M-1, respectively) according to stopped-flow fluorimetry. A gel-based assay showed that LYS228 bound mainly to E. coli PBP3, with weaker binding to PBP1a and PBP1b. Exposing E. coli cells to LYS228 caused filamentation consistent with impaired cell division. No single-step mutants were selected from 12 Enterobacteriaceae strains expressing different classes of ?-lactamases at 8× the MIC of LYS228 (frequency, <2.5 × 10-9). At 4× the MIC, mutants were selected from 2 of 12 strains at frequencies of 1.8 × 10-7 and 4.2 × 10-9 LYS228 MICs were ?2 ?g/ml against all mutants. These frequencies compared favorably to those for meropenem and tigecycline. Mutations decreasing LYS228 susceptibility occurred in ramR and cpxA (Klebsiella pneumoniae) and baeS (E. coli and K. pneumoniae). Susceptibility of E. coli ATCC 25922 to LYS228 decreased 256-fold (MIC, 0.125 to 32 ?g/ml) after 20 serial passages. Mutants accumulated mutations in ftsI (encoding the target, PBP3), baeR, acrD, envZ, sucB, and rfaI These results support the continued development of LYS228, which is currently undergoing phase II clinical trials for complicated intraabdominal infection and complicated urinary tract infection (registered at ClinicalTrials.gov under identifiers NCT03377426 and NCT03354754).

SUBMITTER: Dean CR

PROVIDER: S-EPMC6153799 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Mode of Action of the Monobactam LYS228 and Mechanisms Decreasing <i>In Vitro</i> Susceptibility in Escherichia coli and Klebsiella pneumoniae.

Dean Charles R CR Barkan David T DT Bermingham Alun A Blais Johanne J Casey Fergal F Casarez Anthony A Colvin Richard R Fuller John J Jones Adriana K AK Li Cindy C Lopez Sara S Metzger Louis E LE Mostafavi Mina M Prathapam Ramadevi R Rasper Dita D Reck Folkert F Ruzin Alexey A Shaul Jacob J Shen Xiaoyu X Simmons Robert L RL Skewes-Cox Peter P Takeoka Kenneth T KT Tamrakar Pramila P Uehara Tsuyoshi T Wei Jun-Rong JR

Antimicrobial agents and chemotherapy 20180924 10

The monobactam scaffold is attractive for the development of new agents to treat infections caused by drug-resistant Gram-negative bacteria because it is stable to metallo-β-lactamases (MBLs). However, the clinically used monobactam aztreonam lacks stability to serine β-lactamases (SBLs) that are often coexpressed with MBLs. LYS228 is stable to MBLs and most SBLs. LYS228 bound purified <i>Escherichia coli</i> penicillin binding protein 3 (PBP3) similarly to aztreonam (derived acylation rate/equi ...[more]

PMID: 30061293

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Project description:Antimicrobial resistance genes (ARG), such as extended-spectrum β-lactamase (ESBL) and carbapenemase genes, are commonly carried on plasmids. Plasmids can transmit between bacteria, disseminate globally, and cause clinically important resistance. Therefore, targeting plasmids could reduce ARG prevalence, and restore the efficacy of existing antibiotics. Cobalt complexes possess diverse biological activities, including antimicrobial and anticancer properties. However, their effect on plasmid conjugation has not been explored yet. Here, we assessed the effect of four previously characterised bis(N-picolinamido)cobalt(II) complexes lacking antibacterial activity on plasmid conjugation in Escherichia coli and Klebsiella pneumoniae. Antimicrobial susceptibility testing of these cobalt complexes confirmed the lack of antibacterial activity in E. coli and K. pneumoniae. Liquid broth and solid agar conjugation assays were used to screen the activity of the complexes on four archetypical plasmids in E. coli J53. The cobalt complexes significantly reduced the conjugation of RP4, R6K, and R388 plasmids, but not pKM101, on solid agar in E. coli J53. Owing to their promising activity, the impact of cobalt complexes was tested on the conjugation of fluorescently tagged extended-spectrum β-lactamase encoding pCTgfp plasmid in E. coli and carbapenemase encoding pKpQILgfp plasmid in K. pneumoniae, using flow cytometry. The complexes significantly reduced the conjugation of pKpQILgfp in K. pneumoniae but had no impact on pCTgfp conjugation in E. coli. The cobalt complexes did not have plasmid-curing activity, suggesting that they target conjugation rather than plasmid stability. To our knowledge, this is the first study to report reduced conjugation of clinically relevant plasmids with cobalt complexes. These cobalt complexes are not cytotoxic towards mammalian cells and are not antibacterial, therefore they could be optimised and employed as inhibitors of plasmid conjugation.

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Mode of Action of the Monobactam LYS228 and Mechanisms Decreasing In Vitro Susceptibility in Escherichia coli and Klebsiella pneumoniae.

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Mode of Action of the Monobactam LYS228 and Mechanisms Decreasing <i>In Vitro</i> Susceptibility in Escherichia coli and Klebsiella pneumoniae.

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Mode of Action of the Monobactam LYS228 and Mechanisms Decreasing In Vitro Susceptibility in Escherichia coli and Klebsiella pneumoniae.

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Mode of Action of the Monobactam LYS228 and Mechanisms Decreasing &lt;i&gt;In Vitro&lt;/i&gt; Susceptibility in Escherichia coli and Klebsiella pneumoniae.

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Mode of Action of the Monobactam LYS228 and Mechanisms Decreasing <i>In Vitro</i> Susceptibility in Escherichia coli and Klebsiella pneumoniae.