In vitro activity of sulbactam-durlobactam against colistin-resistant and/or cefiderocol-non-susceptible, carbapenem-resistant Acinetobacter baumannii collected in U.S. hospitals

ABSTRACT

The activity of a novel β-lactamase inhibitor combination, sulbactam-durlobactam (SUL-DUR), was tested against 87 colistin-resistant and/or cefiderocol-non-susceptible carbapenem-resistant Acinetobacter baumannii clinical isolates collected from U.S. hospitals between 2017 and 2019. Among them, 89% and 97% were susceptible to SUL-DUR and imipenem plus SUL-DUR, with MIC₅₀/MIC₉₀ values of 2 µg/mL/8 µg/mL and 1 µg/mL/4 µg/mL, respectively. The presence of amino acid substitutions in penicillin-binding protein 3, including previously reported A515V or T526S, was associated with SUL-DUR non-susceptibility.

INTRODUCTION

Carbapenem-resistant Acinetobacter baumannii causes serious infections, including bacteremia, healthcare-associated pneumonia, and skin and soft tissue infection. Treatment options are extremely limited due to multidrug resistance. Regrettably, short-term mortality from this infection exceeds 20% (1). Sulbactam-durlobactam (SUL-DUR) is a novel β-lactamase inhibitor combination with activity against carbapenem-resistant A. baumannii (2). Sulbactam is a β-lactamase inhibitor that possesses intrinsic activity against A. baumannii through inhibition of penicillin-binding protein 3 (PBP3), an enzyme involved in the synthesis of peptidoglycan, a major component of the cell wall. Durlobactam is a novel diazabicyclooctane β-lactamase inhibitor that, although not directly active against A. baumannii, exhibits potent activity against class A, class C, and class D β-lactamases, including the ADC and OXA carbapenemases produced by carbapenem-resistant A. baumannii (3). SUL-DUR combined with imipenem (IPM)-cilastatin showed promise as a clinical agent in combination with IPM-cilastatin in a phase 3 trial that enrolled patients with hospital-acquired bacterial pneumonia, ventilator-associated bacterial pneumonia, ventilated pneumonia, or bacteremia (4), leading to its approval by the U.S. Food and Drug Administration (FDA) in May of 2023 for treatment of hospital-acquired and ventilator-acquired bacterial pneumonia caused by A. baumannii in adults.

Resistance to SUL-DUR has been associated with presence of class B metallo-β-lactamases and amino acid substitutions in PBP3 (3, 5). The aim of the present study was to determine the activity of SUL-DUR, alone and in combination with IPM against carbapenem-resistant A. baumannii isolates resistant to colistin and/or non-susceptible to cefiderocol, both agents of last resort against this pathogen.

A total of 87 unique colistin-resistant and/or cefiderocol-non-susceptible (as defined by the Clinical and Laboratory Standard Institute (CLSI)’s breakpoints of ≥4 and ≥8 µg/mL, respectively) carbapenem-resistant A. baumannii isolates collected from hospitals across the U.S. between 2017 and 2019 were tested (1). These isolates were obtained through the Study Network of Acinetobacter as a Carbapenem-Resistant Pathogen (SNAP), a prospective, observational carbapenem-resistant A. baumannii cohort study sponsored by the Antibacterial Resistance Leadership Group (ARLG), in which 21 U.S. health systems participated. Colistin-resistant isolates [minimum inhibitory concentration (MIC) ≥4 µg/mL] and cefiderocol-non-susceptible isolates (MIC ≥8 µg/mL) were identified using Sensititre GNX2F (Thermo Fisher, Waltham, MA) and by broth microdilution using iron-depleted, cation-adjusted Mueller-Hinton broth (BD, Sparks Glencoe, MD), respectively. In total, 68 colistin-resistant isolates, 26 cefiderocol-non-susceptible isolates, and 7 isolates that met both criteria were identified. The strain details are provided in the Table S1. The activity of sulbactam, durlobactam, and IPM alone and in different combinations was determined using custom frozen broth microdilution testing plates provided by Entasis Therapeutics Inc. Sulbactam and IPM alone were evaluated using twofold dilutions. For SUL-DUR combination, twofold dilutions of sulbactam were used alongside a constant concentration of 4 µg/mL of durlobactam. For sulbactam and IPM, and SUL-DUR and IPM combination, a 1:1 fixed ratio of sulbactam and IPM was used, with durlobactam set at 4 µg/mL. Escherichia coli ATCC 25922 and A. baumannii NCTC13304 were used as control strains. The FDA susceptibility breakpoint (≤4 µg/mL) was used for SUL-DUR and applied to IPM plus SUL-DUR (https://www.fda.gov/media/167096/download). Whole-genome sequencing was performed on all isolates with Illumina NextSeq or MiSeq technology, and data were queried for sequence types (STs) and β-lactamase genes. Additionally, non-synonymous mutations in mrcA (encoding PBP1a), mrcB (PBP1b), pbpA (PBP2), and ftsI (PBP3) were identified using ATCC 17978 sequences as a reference as previously reported (6). Substitutions present in >90% of isolates were removed from the analysis.

Most isolates (75 of 87) belonged to clonal complex (CC) 2, as defined by the Pasteur Institute MLST scheme. Five isolates belonged to ST499, and the rest belonged to miscellaneous STs. A previously described CC2C sub-lineage (ST2^Pas/ST281^Ox and its single locus variants) was the predominant sub-lineage among CC2 isolates (57 of 75) (1). Overall, 77 of 87 (89%) and 84 of 87 (97%) were susceptible to SUL-DUR and IPM plus SUL-DUR, with MIC₅₀/MIC₉₀ values of 2 µg/mL/8 µg/mL and 1 µg/mL/4 µg/mL, respectively. In comparison, MIC₅₀/MIC₉₀ values of sulbactam alone were 16 µg/mL/64 µg/mL, indicating an 8- to 16-fold reduction of MICs achieved in the presence of durlobactam. Combinations of IPM with sulbactam or durlobactam have only shown two- to fourfold reduction in MIC₅₀/MIC₉₀ (Table 1). For comparison, the MIC₅₀/MIC₉₀ for SUL-DUR against multidrug-resistant A. baumannii isolates from Greece was 4 µg/mL/8 µg/mL, while in Italy, it was 0.5 µg/mL/4 µg/mL. These findings suggest geographical variability in SUL-DUR MIC distribution and underscore the importance of regional and genetic surveillance (7, 8). Of the colistin-resistant isolates, 60 of 68 (88%) and 65 of 68 (96%) were susceptible to SUL-DUR and IPM plus SUL-DUR, with MIC₅₀/MIC₉₀ values of 2 µg/mL/8 µg/mL and 1 µg/mL/4 µg/mL, respectively. Of the cefiderocol-non-susceptible isolates, 22 of 26 (85%) and 25 of 26 (96%) were susceptible to SUL-DUR and IPM plus SUL-DUR, with MIC₅₀/MIC₉₀ values of 4 µg/mL/8 µg/mL and 1 µg/mL/4 µg/mL, respectively. Of the 10 SUL-DUR-non-susceptible isolates, 6 isolates had an MIC of 8 µg/mL, and the addition of IPM lowered the MIC to ≤4 µg/mL. The remaining four isolates had SUL-DUR MICs between 16 and 64 µg/mL, and two each had MICs of 4 and 8 µg/mL with the addition of IPM, respectively (Table S1).

TABLE 1.

In vitro susceptibility of 87 Acinetobacter baumannii isolates from the study

	MIC range (μg/mL)	MIC50 (μg/mL)	MIC90 (μg/mL)
Single agents
SUL	1 to >64	16	64
DUR	64 to >64	>64	>64
IPM	4–64	32	64
Double combinations
SUL-DUR	0.5–64.0	2	8
IPM-DUR	0.5–32.0	8	32
IPM-SUL	2–32	8	16
Triple combination
IPM-SUL-DUR	0.25–8.0	1	4

^a DUR, durlobactam; IPM, imipenem; MIC, minimum inhibitory concentration; SUL, sulbactam.

The following PBP3 predicted amino acid substitutions were identified through whole-genome sequencing across study isolates: Y196S, H370Y, K389E, T511S, A515V, T526S, and F548I. Clinical isolates with A515V or T526S substitutions, which are close to the PBP3 active site, are associated with elevated SUL-DUR MICs in the range of 8–64 µg/mL (6, 9). Among 10 SUL-DUR-non-susceptible isolates, 9 belonged to the CC2C lineage. Two were genomically identical with 0 core genome single-nucleotide polymorphisms (cgSNPs), determined by Snippy v.4.6.0, between them (https://github.com/tseemann/snippy). The rest had a median of 539 cgSNPs (range 79–1944), suggesting genetically diverse isolates. Overall, any PBP3 substitutions were more common in SUL-DUR-non-susceptible isolates [5 of 10 (50%) vs 5 of 77 (6%), P = 0.002 by Fisher’s exact test]. While several substitutions were identified in the other PBPs (PBP1a, PBP1b, and PBP2), they did not seem to correlate with increased SUL-DUR MICs (Table S1). Likewise, we did not observe a correlation between the specific substitutions or OXA carbapenemase and the fold reduction of SUL-DUR MICs by the addition of IPM among the SUL-DUR-non-susceptible isolates. Metallo-β-lactamase genes were not identified among the non-susceptible isolates. In half of SUL-DUR-non-susceptible isolates, we did not identify substitutions in PBP1a, PBP1b, PBP2, and PBP3 which may be associated with SUL-DUR non-susceptibility, suggesting the presence of additional resistance mechanisms. However, the MICs for a majority of non-susceptible isolates were only one dilution higher than those of the susceptibility cut-off value (8 µg/mL vs 4 µg/mL), which may contribute to the lack of a clear genetic distinction. MICs for all antimicrobial agents tested and associated PBP substitutions for individual isolates are summarized in Table S1.

In conclusion, SUL-DUR was active in vitro against a majority of colistin-resistant and/or cefiderocol-non-susceptible carbapenem-resistant A. baumannii clinical isolates from the U.S., with the addition of IPM lowering the median MIC by twofold to fourfold using the method studied. Whether SUL-DUR should be routinely administered in combination with IPM when treating carbapenem-resistant A. baumannii infections remains an area of uncertainty. Finally, half of SUL-DUR non-susceptible isolates lacked known resistance mechanisms, highlighting the need for further investigation (Fig. 1).

Fig 1.

Distruption of minimal inhibitory concentrations (MICs) for sulbactam-durlobactam (SUL-DUR) and imipenem with sulbactam-durlobactam (IPM-SUL-DUR) of colistin-resistant (A) and cefiderocol-non-susceptible (B) isolates. The numbers of isolates with each MIC are listed on top of the histogram bars. Susceptibility breakpoints to SUL-DUR are noted by dashed vertical lines.

Bacterial strains used in this study are available by request through the ARLG (https://arlg.org/laboratory-center-strain-access/).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aac.01258-23.

Supplemental Table S1. aac.01258-23-s0001.xlsx.

Strains used in the study. Sequence type (ST) by the Pasteur Institute Protocol (PasST), the Oxford Protocol (OxST), minimum inhibitory concentration (MIC) of imipenem (IPM), sulbactam (SUL), imipenem plus sulbactam (IPM-SUL), imipenem plus durlobactam (IPM-DUR), sulbactam-durulobactam (SUL-DUR), imipenem plus sulbactam-durlobactam (IPM-SUL-DUR) in µg/mL, susceptibility interpretation of colistin and cefiderocol (FDC), deduced PBP3, PBP1a, PBP1b, and PBP2 substitutions, acquired OXA carbapenemase (pOXA), and intrinsic carbapenemase (cOXA) are listed for each study strain.

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