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-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 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 β-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 μg/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 μg/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).

Keywords: Enterobacteriaceae; LYS228; beta-lactamases; drug resistance mechanisms; mechanisms of action; monobactam.

Copyright © 2018 American Society for Microbiology.

Figures

FIG 1

Chemical structure of LYS228.

FIG 2

(A) Binding of LYS228 or ATM to PBPs, measured qualitatively by gel-based PBP binding assay. (B) Densitometry analysis for PBP binding represents the means from 4 determinations (± standard errors of the means). Both compounds mainly bound PBP3, with lower but detectable binding to PBP1a/b and PBP5/6. ATM bound PBP1a/b with slightly higher affinity than did LYS228. Neither compound showed any detectable binding to PBP2.

FIG 3

Morphology of E. coli cells exposed to LYS228 or control compounds. LYS228 and ATM caused cell filamentation with segregated chromosomal DNA (compare to CIP treatment), suggesting blocked cell division.

FIG 4

Decrease in E. coli NB27001 susceptibility to LYS228 and comparator antibiotics over 20 serial passages. Passage 0 represents the initial LYS228 MIC for NB27001.