Molecular mechanisms of sulbactam antibacterial activity and resistance determinants in Acinetobacter baumannii

Abstract

Sulbactam is a class A β-lactamase inhibitor with intrinsic whole-cell activity against certain bacterial species, including Acinetobacter baumannii. The clinical use of sulbactam for A. baumannii infections is of interest due to increasing multidrug resistance in this pathogen. However, the molecular drivers of its antibacterial activity and resistance determinants have yet to be precisely defined. Here we show that the antibacterial activities of sulbactam vary widely across contemporary A. baumannii clinical isolates and are mediated through inhibition of the penicillin-binding proteins (PBPs) PBP1 and PBP3, with very low frequency of resistance; the rare pbp3 mutants with high levels of resistance to sulbactam are attenuated in fitness. These results support further investigation of the potential clinical utility of sulbactam.

FIG 1

Sulbactam binding to PBP1 and PBP3 but not PBP2 in A. baumannii. Increasing amounts of compounds of interest were incubated with membrane proteins harvested from A. baumannii ATCC 17978 in a standard, gel-based, Bocillin FL competition assay. Arrows, PBPs 1a/b, PBP2, and PBP3. There is no pbp1c gene in any of the published A. baumannii genomes. It is likely that the band corresponding to this protein, as documented for other Gram-negative orthologs, is a proteolytic fragment or results from an alternate start site for PBP1a or PBP1b. MWM, molecular weight markers.

FIG 2

Fluorescence microscopic images of cells after 3-h exposure to 0.5× MIC levels of antimicrobials. (A to E) A. baumannii ARC2058. (A) No drug. (B) Sulbactam (0.5 μg/ml). (C) Ceftazidime (4 μg/ml). (D) Aztreonam (16 μg/ml). (E) Meropenem (0.125 μg/ml). (F) Sulbactam-resistant ATCC 17978 pbp3 S395F mutant, without treatment. (G and H) ARC2058 pbp3 S390T mutant, without treatment (G) and after exposure to sulbactam at 16 μg/ml (H). Bars, 5 μm. Similar results were obtained with ATCC 17978 (data not shown).

FIG 3

Sulbactam-resistant A. baumannii attenuation in growth. Wild-type or sulbactam-resistant mutant strains were incubated at 37°C with shaking, either in MHB-II (A) or in freshly harvested, pooled, mouse blood in a 5% CO₂ incubator (B). Samples were removed at the indicated times, and bacterial proliferation was measured by performing colony counts of 2-fold serial dilutions of these samples on blood agar, after overnight incubation at 37°C. Results shown are averages of at least three independent experiments.