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 penicillin binding protein 3 (PBP3) similarly to aztreonam (derived acylation rate/equilibrium dissociation constant [/ ] of 367,504 s M and 409,229 s M, respectively) according to stopped-flow fluorimetry. A gel-based assay showed that LYS228 bound mainly to PBP3, with weaker binding to PBP1a and PBP1b. Exposing cells to LYS228 caused filamentation consistent with impaired cell division. No single-step mutants were selected from 12 strains expressing different classes of β-lactamases at 8× the MIC of LYS228 (frequency,<2.5 × 10). At 4× the MIC, mutants were selected from 2 of 12 strains at frequencies of 1.8 × 10 and 4.2 × 10 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 and () and ( and ). Susceptibility of ATCC 25922 to LYS228 decreased 256-fold (MIC, 0.125 to 32 μg/ml) after 20 serial passages. Mutants accumulated mutations in (encoding the target, PBP3),, ,, , and 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).

Impact of InducibleblaDHA-1on Susceptibility of Klebsiella pneumoniae Clinical Isolates to LYS228 and Identification of ChromosomalmplandampDMutations Mediating Upregulation of Plasmid-BorneblaDHA-1Expression

“…The MIC of LYS228 increased to 8 and 32 g/ml for these mutants (NB29289-CDK0033 and CDK0034) (Table 1), and again LYS228 susceptibility was restored by the addition of avibactam (Table 1). It should be noted that no mpl mutations were isolated from several non-bla DHA-1 K. pneumoniae isolates during single-step selections described in an accompanying report (33), strengthening the association of mpl mutations with bla DHA-1 . Mpl is UDP-N-acetylmuramate:L-alanyl-␥-D-glutamyl-meso-diaminopimelate ligase, involved in peptidoglycan recycling (34,35), and its mutational loss may induce expression of bla DHA-1 via changes in peptidoglycan intermediates sensed by the plasmid-encoded AmpR regulator.…”

Stepwise evolution and convergent recombination underlie the global dissemination of carbapenemase-producingEscherichia coli

“…To our knowledge these modifications are the only mutations contributing to β-lactams reported in natural E. coli isolates 19,25 . Nevertheless, the A413V mutation found in association with three types of insertions, has recently been selected during serial passages of E. coli in the presence of aztreonam and shown to lead to a four-fold decreased susceptibility to this antibiotic 38 . This result is in agreement with our prediction that this mutation might contribute to resistance to β-lactams of isolates carrying YRIK, YTIP or YRIP insertions as we have shown for the two SNPs associated with the YRIN duplication (Fig.…”

“…ftsI mutations were also found to be selected during in vitro evolution in the presence of ertapenem but not meropenem 43 . However, other β-lactams like aztreonam might also have contributed to the selection of these combinations of mutations 38 .…”

Critical analysis of antibacterial agents in clinical development

“…Additionally, it has been modified to resist hydrolysis by most serine β lactamases in Enterobacteriaceae 68 . Cross resistance exists with aztreonam when non β lactamase mechanisms are involved (for example, upregulated efflux and/or porin downregulation, or mutations in PBP3 and the envelope stress response system) 69 .…”