Sulbactam combined with the Novel β-lactamase Inhibitor ETX2514 for the Treatment of Multidrug-resistant Acinetobacter baumannii Infections

Background: Multidrug resistant (MDR) Acinetobacter baumannii infections are of great concern due to high mortality rates and limited number of treatment options. β-lactamase (BL) expression, especially Class D, is an important resistance mechanism in this organism. The novel BL inhibitor ETX2514 has potent activity against Class A, C and D serine BLs. The MIC₉₀ of sulbactam (SUL) in the presence of ETX2514 is 4 mg/L against a large, globally diverse set of MDR A. baumannii clinical isolates from 2014. In this study, we investigate the mechanism of synergy of this combination alone or in the presence of imipenem (IPM) or meropenem (MEM), whose spectra of target inhibition vary across bacterial species

Methods: MICs were performed according to CLSI guidelines. The frequency of resistance (FOR) to SUL/ETX2514 was determined in several clinical isolates of A. baumannii.  Resistant mutants were analyzed by whole genome sequencing (WGS). Morphological changes were examined by microscopy. PBP acylation rates were determined by competition with BOCILLIN FL in fluorescence polarization assays.

Results: The MIC₅₀ and MIC₉₀ of relevant combinations against 598 contemporary isolates of A. baumannii are shown below.

Compound(s)	MIC50	MIC90
SUL	1	>32
IPM	32	>32
MEM	32	>32
SUL/ETX2514*	1	4
IPM/ETX2514*	4	16
IPM/SUL*	32	>32
IPM/SUL*/ETX2514*	≤0.03	≤0.03
MEM/SUL*/ETX2514*	≤0.03	4

*held constant at 4 mg/L

SUL-treated A. baumannii strains had elongated cell morphologies which became spherical when ETX2514 was added, a phenotype associated with PBP2 inhibition in Escherichia coli. The FOR to SUL/ETX2514 at 4X MIC was 7.6 x 10^-10. Resistance in A. baumannii mapped to residues S390T, S395F or F548C in PBP3 or to mutations in tRNA synthetase genes (aspS and gltX). The latter are associated with resistance to PBP2 inhibitors in E. coli. Purified mutant PBP3 proteins had reduced affinity for SUL and variable affinity for IPM and MEM in BOCILLIN competition assays.

Conclusion: Resistance mapping and morphological changes suggest that, in addition to BL inhibition, ETX2514 enhances antibacterial activity through PBP2 inhibition in A. baumannii. The remarkable synergy observed with the triple combination IMP/SUL/ETX2514 likely reflects superior antibacterial potency achieved through a combination of BL inhibition and expanded PBP inhibition.