Activity of Sulbactam-Durlobactam and Comparators Against a National Collection of Carbapenem-Resistant Acinetobacter baumannii Isolates From Greece

Background Acinetobacter baumannii is a leading cause of healthcare-associated infections worldwide, due to both its persistence in the hospital setting and ability to acquire high levels of antibiotic resistance. Carbapenem-resistant A. baumannii isolates (CRAB) limit the activity of current antimicrobial regimens and new alternatives or adjuncts to traditional antibiotics are urgently needed. Durlobactam is a novel broad-spectrum inhibitor of serine-type β-lactamases that restores sulbactam (SUL) activity against A. baumannii. The sulbactam-durlobactam (SD) combination has recently completed Phase 3 testing in the global ATTACK trial. Objectives The aim of this study is to evaluate the in vitro activity of SD versus comparators against a representative nationwide collection of CRAB isolates. Methods One hundred ninety CRAB isolates were collected from clinical samples of patients hospitalized in 11 hospitals throughout Greece during 2015. In vitro activities of SD and comparators (SUL alone, amikacin, minocycline, imipenem, meropenem, colistin, SD and imipenem combined with SD) were determined by broth microdilution. Results Durlobactam restored sulbactam activity against the majority of the strains tested, with SD exhibiting the lowest MIC90 (8 μg/ml) relative to the other single comparators tested; 87.9% of the isolates had SD MICs ≤4/4 µg/ml. The most active comparator was colistin (MIC90 = 16 μg/ml). The addition of imipenem further lowered the MIC90 of SD by one two-fold dilution. Conclusions This study demonstrated the potential utility of SD for the treatment of infections caused by A. baumannii. If its clinical efficacy is confirmed, SD may be an important therapeutic option for CRAB infections.


INTRODUCTION
Infections caused by multidrug-resistant (MDR) A. baumannii pose a serious threat to global health. These infections include ventilator-associated pneumonia, bacteremia, complicated urinary tract infections and skin and soft tissue infections, in both healthy and immuno-compromised individuals (Lee et al., 2017). The mortality rates of these infections reach 33%, and carbapenem resistance was associated with a greater risk of death (pooled odds ratio 2.22, 95% CI 1.66-2.98) [European Centre for Disease Prevention and Control (ECDC), 2013; Lemos et al., 2014]. A recent analysis of the global prevalence of antibiotic resistance in A. baumannii infections found a prevalence of resistance to imipenem of 73.9-77.8%, which represented a dramatic increase since 2005 (Xie et al., 2018).
The epidemiological status of carbapenem-resistant A. baumannii (CRAB) in Greece is defined as endemic situation, with most hospitals repeatedly facing cases admitted from endemic sources [European Centre for Disease Prevention and Control (ECDC), 2013]. The resistance to carbapenems in A. baumannii precludes the use of b-lactam therapies; the main mechanism responsible is the production of oxacillinases (class D b-lactamases), with the most frequent ones being OXA-23, OXA-24/40 and OXA-58 (D'Arezzo et al., 2010;Gogou et al., 2011;Schleicher et al., 2013). Among the limited b-lactams that retain a degree of activity against A. baumannii is sulbactam (SUL), a semisynthetic penicillanic acid, which is a first-generation b-lactamase inhibitor, with limited activity against class A serine b-lactamases Shapiro et al., 2021). SUL inhibits A. baumannii enzymes that are involved in bacterial peptidoglycan synthesis, such as PBP1a, PBP1b and PBP3 (Higgins et al., 2014). However, due to its susceptibility to degradation by a variety of acquired or upregulated b-lactamases, SUL has limited clinical utility against infections caused by CRAB (Penwell et al., 2015).
A promising new therapeutic option for CRAB is the combination of sulbactam with durlobactam (SD), a new member of the diazabicyclooctane class of b-lactamase inhibitors, with broad spectrum activity against Ambler class A, C and D serine blactamases, resulting in the restoration of the susceptibility of CRAB isolates to b-lactams (Durand-Reville et al., 2017). Durlobactam (DUR) restores SUL susceptibility of A. baumannii strains overexpressing individual b-lactamases Shapiro et al., 2021) as well as diverse clinical isolates, in international, contemporary surveillance studies (McLeod et al., 2020;Yang et al. 2020;Nodari et al. 2021). SD is currently in late-stage development for the treatment of infections caused by Acinetobacter spp.
The aim of this study was to examine the in vitro potency of the SD combination on a collection of previously characterized, non-duplicate isolates of CRAB from Greece harboring acquired b-lactamases.

Next Generation Sequencing of Isolates With Elevated SD MICs
Three isolates with SD MICs >8/4 µg/ml were analyzed by next Generation Sequencing. Genomic DNA was purified from the isolates using a Sigma-Aldrich GenElute bacterial genomic DNA kit. Genomic libraries were assembled using a Nextera XT library preparation kit and sequenced using an Illumina MiSeq system with 300-bp paired-end reads and a coverage of ≥ 50X. Assembly and analysis of the whole genome sequencing was performed using CLC Genomics Workbench v21.0.3 (Qiagen, Germantown, MD). Paired Fastq files were processed and analyzed as follows: raw reads were trimmed of any remaining barcode sequences as well as trimmed for quality. Reads were then de novo assembled using fraction length=0.8 and similarity fraction=0.9 using default mismatch/insertion/deletion costs. Consensus sequences were extracted and contigs greater than 500bp were assembled. B-lactamase genes for each strain were identified by BLAST against a local b-lactamase database. Additionally, mutations in efflux, permeation, and PBP proteins were identified by BLAST analysis against the A. baumannii ATCC 17978 reference strain. The genomic sequences of the three strains tested were deposited at http:// www.ncbi.nlm.nih.gov/bioproject/781741.
Of the isolates, 87.9% had SD MICs of ≤4/4 µg/ml and only three isolates had SD MIC >8 µg/ml. These three isolates were submitted to NGS sequencing. The genomic characteristics of these isolates are shown in Table 3. In brief, they belonged to three different MLST types (ST-1834, SD MIC 16 mg/ml; ST-1294 SD MIC >64 mg/ml; and ST-425, SD MIC 16 mg/ml). They all carried bla OXA-23 and bla OXA-66 and all encoded the same A515V variant of the PBP3 gene that likely confers resistance to SUL, considering its proximity to the SUL binding site (Papp-Wallace et al., 2012). The SID MIC for both ST-1834 and ST-425 isolates was 4 mg/ml (i.e. addition of IMP helped reduce the SD MIC), while the ST-1294 isolate had SD MIC of > 64 mg/ml and SID MIC 64/64/128 mg/ml. The latter isolate also harbored the NDM metallo-b-lactamase gene, which DUR does not inhibit.

DISCUSSION
The most prevalent mechanism of carbapenem resistance among A. baumannii is associated with carbapenem-hydrolysing enzymes that belong to Ambler class D and B b-lactamases (Jeon et al., 2015;Lee et al., 2017;Wong et al., 2017;Lötsch et al., 2020). The rapid rise of carbapenem resistance among A. baumannii isolates limits the available therapeutic options and poses a serious need for new antimicrobial agents. In particular, according to 2019 data from the European Antimicrobial Resistance Surveillance Network (EARS-Net), nearly a third of invasive Acinetobacter spp. isolates in the EU/EEA are already resistant to carbapenems [European Centre for Disease Prevention and Control (ECDC), 2019; Lötsch et al., 2020]. Carbapenem resistance rates are higher than 50% in southern and eastern European countries [European Centre for Disease Prevention and Control (ECDC), 2019], while in Greece, according to data from the Hellenic CDC, IMP resistance rates are currently exceeding 90% (WHONET). Of note, in our representative countrywide collection, CRAB isolates exhibited particularly high levels of resistance to last-line antimicrobials in addition to carbapenems, including AMK, SUL, MIN and COL.
Among the few therapeutic options that show efficacy against CRAB isolates is COL, used both as monotherapy or in combination with other antimicrobials. Still, while COL is a key drug, there are concerns, not only about its toxicity profiles, but also its rising resistance rates (Viehman et al., 2014). A metaanalysis on the prevalence of A. baumanni antimicrobial resistance worldwide from 2000 to 2017, showed that the overall global resistance rate reaches 11.2% (Pormohammad et al., 2020). Herein, COL showed a much higher resistance rate of 32.1% and among isolates, collected from blood cultures, the rate reached 36%.
The carbapenem resistance problem of A. baumannii can be overcome by the use of expanded-spectrum serine b-lactamase inhibitors, which may inhibit class A, C or D b-lactamases, resulting in restoration of b-lactam activity. SD is a promising combination and its spectrum of activity can address MDR A. baumannii. In our study, the addition of DUR at a fixed concentration of 4 µg/ml lowered the MIC 50 and MIC 90 of SUL from 64 and >64 to 4 and 8 mg/ml, respectively, except for the isolate that encoded metallo-b-lactamase. The MIC 90 of SD was considerably lower than the MIC 90 of carbapenems, MIN and AMK and also lower from that of COL [16 mg/ml]. 88.5% of non-susceptible to COL isolates had SD MICs ≤ 4 mg/ml.
A. baumannii isolates belonging to IC1 showed generally more sensitive profiles compared to IC2, concerning SUL, COL and carbapenems. For MIN, the resistance rates between IC1 and IC2 isolates were 2.8% and 71.2%, respectively. There was no difference on the MICs of SD and SID among the isolates of the two clonal lineages. For both IC2 and IC2 isolates, MIC 50/90 of SD and SID were at 4/4 and 2/4, respectively. Compared to other studies on the activity of SD, our collection of isolates presented higher resistance rates and MICs for the comparators, as well as for SD. In particular, both the SD MIC 50 and MIC 90 of the strains in the present study exceeded by one to three-fold the respective values of those reported in international studies (McLeod et al., 2020;Yang et al., 2020;Nodari et al., 2021), indicating the presence of less susceptible strains in Greek hospitals. COL MICs were also considerably lower in three of those studies (MIC 90 1 mg/ml in references Higgins et al., 2004;Pournaras et al., 2017;, with only one study from China reporting COL MIC 90 128 mg/ml (Durand-Réville et al., 2017). Interestingly, the addition of IMP to SD lowered its MIC90 by one twofold dilution.
Our study clearly showed that SD had excellent in vitro activity against CRAB isolates that were highly resistant to IMP, MER, AMK, MIN and COL. In addition, SD showed favorable clinical efficacy and safety in a recently completed, global phase 3 study (Entasis Therapeutics, 2019), https://investors.entasistx.com/ news-releases/news-release-details/entasis-therapeuticsannounces-positive-topline-results). If approved this combination may provide an important therapeutic option for infections due to MDR A. baumannii, including CRAB.
Part of this work was presented at the 31st European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Online, 9-12 July 2021.

DATA AVAILABILTY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

FUNDING
The research being submitted received a grant from ENTASIS Therapeutics. This grant did not include open access publication fees, which will be paid by the research account of our Laboratory in our institution (National and Kapodistrian University of Athens Special Account for Research Grants).

ACKNOWLEDGMENTS
We thank Samir Moussa of Entasis Therapeutics for next generation sequencing and analysis.