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. 2020 Mar 24;64(4):e02534-19. doi: 10.1128/AAC.02534-19

In Vitro Activity of Sulbactam-Durlobactam against Acinetobacter baumannii-calcoaceticus Complex Isolates Collected Globally in 2016 and 2017

Sarah M McLeod a,✉, Samir H Moussa a, Meredith A Hackel b, Alita A Miller a
PMCID: PMC7179289  PMID: 31988095

Acinetobacter baumannii-calcoaceticus complex (ABC) organisms cause severe infections that are difficult to treat due to preexisting antibiotic resistance. Sulbactam-durlobactam (formerly sulbactam-ETX2514) (SUL-DUR) is a β-lactam–β-lactamase inhibitor combination antibiotic designed to treat serious infections caused by ABC organisms, including multidrug-resistant (MDR) strains. The in vitro antibacterial activities of SUL-DUR and comparator agents were determined by broth microdilution against 1,722 clinical isolates of ABC organisms collected in 2016 and 2017 from 31 countries across Asia/South Pacific, Europe, Latin America, the Middle East, and North America.

KEYWORDS: Acinetobacter, ETX2514, diazabicyclooctane, durlobactam, sulbactam, surveillance studies

ABSTRACT

Acinetobacter baumannii-calcoaceticus complex (ABC) organisms cause severe infections that are difficult to treat due to preexisting antibiotic resistance. Sulbactam-durlobactam (formerly sulbactam-ETX2514) (SUL-DUR) is a β-lactam–β-lactamase inhibitor combination antibiotic designed to treat serious infections caused by ABC organisms, including multidrug-resistant (MDR) strains. The in vitro antibacterial activities of SUL-DUR and comparator agents were determined by broth microdilution against 1,722 clinical isolates of ABC organisms collected in 2016 and 2017 from 31 countries across Asia/South Pacific, Europe, Latin America, the Middle East, and North America. Over 50% of these isolates were resistant to carbapenems. Against this collection of global isolates, SUL-DUR had a MIC50/MIC90 of 1/2 μg/ml compared to a MIC50/MIC90 of 8/64 μg/ml for sulbactam alone. This level of activity was found to be consistent across organisms, regions, sources of infection, and subsets of resistance phenotypes, including MDR and extensively drug-resistant isolates. The SUL-DUR activity was superior to those of the tested comparators, with only colistin having similar potency. Whole-genome sequencing of the 39 isolates (2.3%) with a SUL-DUR MIC of >4 μg/ml revealed that these strains encoded either the metallo-β-lactamase NDM-1, which durlobactam does not inhibit, or single amino acid substitutions near the active site of penicillin binding protein 3 (PBP3), the primary target of sulbactam. In summary, SUL-DUR demonstrated potent antibacterial activity against recent, geographically diverse clinical isolates of ABC organisms, including MDR isolates.

INTRODUCTION

Acinetobacter baumannii can cause severe nosocomial infections associated with high mortality rates and is increasingly being reported as extensively drug-resistant (XDR) in many parts of the world (1, 2). One of the most alarming trends is the wide-spread acquisition of class D β-lactamases that confer resistance to carbapenems in clinical isolates of A. baumannii (2–6). Other currently available treatment options have unacceptable toxicity profiles or may be ineffective due to their poor pharmacokinetic properties or preexisting resistance (1).

A combination of sulbactam and durlobactam (also known as ETX2514) (SUL-DUR) is currently in clinical development for the treatment of Acinetobacter baumannii-calcoaceticus complex (ABC) infections (6). Sulbactam is a β-lactam Ambler class A β-lactamase inhibitor (BLI) that also has intrinsic antibacterial activity against Acinetobacter spp., due primarily to inhibition of penicillin binding proteins 1 and 3 (PBP1 and PBP3, respectively), which are essential components of cell wall synthesis (7). However, degradation of sulbactam by a variety of β-lactamases present in most ABC isolates limits its clinical use (6, 8, 9). Durlobactam is a non-β-lactam BLI which has a modified diazabicyclooctane (DBO) scaffold with an extended spectrum of activity compared to those of other DBO inhibitors. Durlobactam inhibits a broad range of class D β-lactamases and has notably more potent inhibition of class A and C β-lactamases (6, 10). While durlobactam does not demonstrate antibacterial activity alone against ABC organisms, it does have intrinsic activity against certain species of Enterobacterales, due to inhibition of PBP2 in these organisms (6).

This study reports the in vitro antibacterial activities of SUL-DUR and comparator antimicrobial agents against contemporary clinical isolates of the ABC complex (A. baumannii, Acinetobacter pittii, Acinetobacter nosocomialis, and Acinetobacter calcoaceticus) collected globally in 2016 and 2017. The study evaluated 1,722 ABC isolates from community- and hospital-associated infection sources in 31 countries across Europe, North America, Latin America, the Middle East, and Asia and the South Pacific. Isolates with reduced susceptibility to SUL-DUR were subjected to whole-genome sequencing to identify the molecular drivers of SUL-DUR resistance.

RESULTS

In vitro activity of SUL-DUR against global clinical ABC isolates.

A surveillance study was conducted to assess the in vitro activity of SUL-DUR and comparator agents against a collection of 1,722 ABC isolates obtained during 2016 (n = 843) and 2017 (n = 879) from 209 medical centers in 31 countries around the world. Only one isolate per patient was collected from the following infection sources: bloodstream (13.9%), intra-abdominal (3.8%), respiratory tract (61.2%), urinary tract (18.3%), skin and soft tissue (0.8%), and other or unknown sources (2.0%). Isolates were collected from Europe (41.4%), North America (United States only) (29.7%), Latin America (15.2%), Asia/South Pacific (12.8%), and the Middle East (Israel only) (0.9%). Since A. baumannii is the most clinically relevant and antibiotic-resistant species of the ABC complex, this collection was predominantly A. baumannii isolates (82.5%). This collection contained 13.5% A. pittii, 3.5% A. nosocomialis, and 0.6% A. calcoaceticus isolates, reflecting their lower levels of prevalence (11, 12).

Against all 1,722 ABC isolates, the addition of durlobactam to sulbactam lowered the MIC90 compared to that of sulbactam alone by 32-fold, from 64 μg/ml to 2 μg/ml (Tables 1 and 2). Over half of these isolates were nonsusceptible to carbapenems, with only 47.4% and 46.0% susceptible to imipenem and meropenem, respectively. The most active compound against this set of isolates was colistin, with 95.3% susceptibility (MIC90 of 1 μg/ml), followed by minocycline, with 80.4% susceptibility (MIC90 of 16 μg/ml), based on current CLSI breakpoint criteria (13). However, the colistin susceptibility results must be interpreted with caution, as susceptibility breakpoint criteria are not recognized for A. baumannii by the FDA (14). The MIC90 of tigecycline was 2 μg/ml; however, the overall susceptibility to this agent cannot be defined because there are no approved breakpoints for tigecycline for the treatment of Acinetobacter spp.

TABLE 1.

In vitro activities of sulbactam-durlobactam and comparator antimicrobial agents tested against 1,722 clinical isolates of ABC collected globally in 2016 and 2017

Geographic region, category (no. of isolates) Antimicrobial agent(s) MIC (μg/ml)
 % susceptiblea
MIC50 MIC90 Range
Global, all (1,722) Sulbactam-durlobactam 1 2 ≤0.03 to >64 NA
Sulbactam 8 64 0.25 to >64 NA
Cefepime 16 >16 ≤0.12 to >16 41.3
Imipenem 16 64 0.06 to >64 47.4
Meropenem 16 >64 0.06 to >64 46.0
Amikacin 4 >64 ≤0.5 to >64 57.0
Ciprofloxacin >4 >4 ≤0.12 to >4 40.9
Colistin 0.5 1 ≤0.25 to >8 95.3
Minocycline 0.5 16 ≤0.12 to >16 80.4
Tigecycline 0.5 2 ≤0.015 to 32 NA
Global, A. baumannii (1,420) Sulbactam-durlobactam 1 4 ≤0.03 to >64 NA
Sulbactam 16 64 0.25 to >64 NA
Cefepime >16 >16 ≤0.12 to >16 31.2
Imipenem 32 64 0.06 to >64 37.0
Meropenem 32 >64 0.06 to >64 35.6
Amikacin 32 >64 ≤0.5 to >64 48.7
Ciprofloxacin >4 >4 ≤0.12 to >4 30.1
Colistin 0.5 1 ≤0.25 to >8 94.4
Minocycline 1 16 ≤0.12 to >16 76.3
Tigecycline 0.5 2 ≤0.015 to 32 NA
Global, A. calcoaceticus (10) Sulbactam-durlobactam 0.5 1 0.12 to 1 NA
Sulbactam 2 4 1 to 4 NA
Cefepime 4 8 4 to 8 100
Imipenem 0.12 0.25 0.12 to 0.25 100
Meropenem 0.25 0.5 0.12 to 0.5 100
Amikacin ≤0.5 1 ≤0.5 to 2 100
Ciprofloxacin ≤0.12 0.25 ≤0.12 to 0.25 100
Colistin 0.5 1 ≤0.25 to 1 100
Minocycline ≤0.12 ≤0.12 ≤0.12 to ≤0.12 100
Tigecycline 0.06 0.12 0.06 to 0.12 NA
Global, A. nosocomialis (60) Sulbactam-durlobactam 0.5 1 0.12 to 4 NA
Sulbactam 2 8 0.5 to 64 NA
Cefepime 2 >16 1 to >16 85.0
Imipenem 0.25 0.25 0.06 to >64 93.3
Meropenem 0.25 1 0.12 to 64 93.3
Amikacin 2 16 ≤0.5 to >64 91.7
Ciprofloxacin 0.25 2 ≤0.12 to >4 85.0
Colistin 0.5 2 ≤0.25 to >8 98.3
Minocycline ≤0.12 0.5 ≤0.12 to 8 98.3
Tigecycline 0.12 1 0.03 to 2 NA
Global, A. pittii (232) Sulbactam-durlobactam 0.5 2 0.12 to 4 NA
Sulbactam 2 4 0.5 to 64 NA
Cefepime 4 8 0.25 to >16 90.1
Imipenem 0.25 0.25 0.12 to 64 96.6
Meropenem 0.5 1 0.06 to 64 95.7
Amikacin 1 2 ≤0.5 to >64 97.4
Ciprofloxacin ≤0.12 0.5 ≤0.12 to >4 93.5
Colistin 0.5 1 ≤0.25 to 2 100
Minocycline ≤0.12 0.25 ≤0.12 to 4 100
Tigecycline 0.12 0.5 0.03 to 2 NA
Asia/South Pacific, all (221) Sulbactam-durlobactam 1 2 0.06 to 64 NA
Sulbactam 32 64 0.5 to >64 NA
Cefepime >16 >16 0.5 to >16 30.3
Imipenem 32 64 0.06 to >64 31.2
Meropenem 64 >64 0.13 to >64 31.7
Amikacin >64 >64 ≤0.5 to >64 42.1
Ciprofloxacin >4 >4 ≤0.12 to >4 30.3
Colistin 0.5 1 ≤0.25 to >8 97.3
Minocycline 2 8 ≤0.12 to >16 77.8
Tigecycline 1 2 0.03 to 8 NA
Europe, all (713) Sulbactam-durlobactam 1 4 ≤0.03 to 64 NA
Sulbactam 8 64 0.25 to >64 NA
Cefepime 16 >16 ≤0.12 to >16 43.8
Imipenem 16 64 0.06 to >64 47.8
Meropenem 8 >64 0.06 to >64 47.5
Amikacin 4 >64 ≤0.5 to >64 57.5
Ciprofloxacin >4 >4 ≤0.12 to >4 41.4
Colistin 0.5 1 ≤0.25 to >8 93.4
Minocycline 0.5 16 ≤0.12 to >16 74.1
Tigecycline 0.5 2 0.03 to 8 NA
Latin America, all (262) Sulbactam-durlobactam 1 4 0.12 to >64 NA
Sulbactam 16 64 0.5 to >64 NA
Cefepime >16 >16 0.5 to >16 23.3
Imipenem 32 >64 0.06 to >64 26.7
Meropenem 64 >64 0.12 to >64 25.6
Amikacin 32 >64 ≤0.5 to >64 37.4
Ciprofloxacin >4 >4 ≤0.12 to >4 22.5
Colistin 0.5 1 ≤0.25 to >8 98.9
Minocycline 0.5 8 ≤0.12 to >16 87.4
Tigecycline 0.5 2 ≤0.015 to 4 NA
Middle East (Israel), all (15) Sulbactam-durlobactam 1 2 0.25 to 2 NA
Sulbactam 8 16 2 to 32 NA
Cefepime >16 >16 2 to >16 6.7
Imipenem 16 32 0.25 to 64 26.7
Meropenem 32 64 0.25 to 64 26.7
Amikacin 32 64 1 to >64 20.0
Ciprofloxacin >4 >4 0.25 to >4 6.7
Colistin 0.5 1 0.5 to 1 100
Minocycline 0.5 16 ≤0.12 to 16 86.7
Tigecycline 1 1 0.12 to 2 NA
North America (USA), all (511) Sulbactam-durlobactam 1 2 ≤0.03 to 8 NA
Sulbactam 4 32 0.25 to >64 NA
Cefepime 8 >16 0.5 to >16 53.0
Imipenem 0.25 64 0.06 to >64 65.0
Meropenem 1 >64 0.12 to >64 61.2
Amikacin 2 >64 ≤0.5 to >64 74.0
Ciprofloxacin 0.5 >4 ≤0.12 to >4 55.4
Colistin 0.5 1 ≤0.25 to >8 95.1
Minocycline 0.25 8 ≤0.12 to >16 86.7
Tigecycline 0.5 2 0.03 to 32 NA
Global, bloodstream infections (238) Sulbactam-durlobactam 1 4 0.06 to 8 NA
Sulbactam 4 32 0.5 to >64 NA
Cefepime 8 >16 0.5 to >16 53.8
Imipenem 0.25 64 0.06 to >64 59.7
Meropenem 1 >64 0.12 to >64 58.8
Amikacin 2 >64 ≤0.5 to >64 67.6
Ciprofloxacin 0.5 >4 ≤0.12 to >4 55.0
Colistin 0.5 1 ≤0.25 to >8 95.0
Minocycline 0.25 16 ≤0.12 to >16 84.0
Tigecycline 0.25 2 0.03 to 4 NA
Global, respiratory tract infections (1,056) Sulbactam-durlobactam 1 2 ≤0.03 to >64 NA
Sulbactam 8 64 0.25 to >64 NA
Cefepime >16 >16 ≤0.12 to >16 36.0
Imipenem 32 64 0.06 to >64 41.2
Meropenem 32 >64 0.06 to >64 39.5
Amikacin 16 >64 ≤0.5 to >64 50.8
Ciprofloxacin >4 >4 ≤0.12 to >4 36.0
Colistin 0.5 1 ≤0.25 to >8 94.2
Minocycline 1 16 ≤0.12 to >16 78.3
Tigecycline 0.5 2 0.03 to 32 NA
Global, urinary tract infections (316) Sulbactam-durlobactam 1 2 0.06 to >64 NA
Sulbactam 4 32 0.25 to >64 NA
Cefepime 16 >16 0.25 to >16 49.7
Imipenem 0.5 64 0.06 to >64 58.5
Meropenem 1 >64 0.06 to >64 57.6
Amikacin 2 >64 ≤0.5 to >64 69.3
Ciprofloxacin >4 >4 ≤0.12 to >4 47.5
Colistin 0.5 1 ≤0.25 to >8 97.8
Minocycline 0.25 16 ≤0.12 to >16 83.2
Tigecycline 0.5 2 0.03 to 8 NA
Global, intra-abdominal infections (65) Sulbactam-durlobactam 1 2 0.12 to 8 NA
Sulbactam 16 32 1 to >64 NA
Cefepime >16 >16 1 to >16 32.3
Imipenem 32 64 0.12 to >64 35.4
Meropenem 32 >64 0.12 to >64 35.4
Amikacin 32 >64 ≤0.5 to >64 47.7
Ciprofloxacin >4 >4 ≤0.12 to >4 29.2
Colistin 0.5 1 ≤0.25 to >8 98.5
Minocycline 0.5 8 ≤0.12 to 16 83.1
Tigecycline 0.5 2 0.03 to 4 NA
Global, skin and soft tissue infections (14) Sulbactam-durlobactam 0.5 1 0.5 to 1 NA
Sulbactam 2 32 0.5 to 64 NA
Cefepime 4 >16 1 to >16 71.4
Imipenem 0.25 1 0.12 to 64 85.7
Meropenem 0.25 32 0.12 to >64 85.7
Amikacin 2 >64 ≤0.5 to >64 85.7
Ciprofloxacin ≤0.12 >4 ≤0.12 to >4 71.4
Colistin 0.5 1 ≤0.25 to 1 100
Minocycline 0.25 0.5 ≤0.12 to 4 100
Tigecycline 0.25 0.5 ≤0.015 to 1 NA
Global, other, and unknown sources of infections (33) Sulbactam-durlobactam 1 2 0.12 to 32 NA
Sulbactam 4 32 0.5 to 64 NA
Cefepime 16 >16 0.5 to >16 48.5
Imipenem 1 64 0.12 to >64 57.6
Meropenem 1 >64 0.12 to >64 57.6
Amikacin 4 >64 1 to >64 67.7
Ciprofloxacin >4 >4 ≤0.12 to >4 45.5
Colistin 0.5 1 ≤0.25 to 1 100
Minocycline 0.25 8 ≤0.12 to 16 81.8
Tigecycline 0.25 1 0.06 to 2 NA
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a

Percentage of isolates susceptible to antimicrobial agent according to CLSI M100-S30 (13). NA, no available breakpoint.

TABLE 2.

MIC cumulative frequency distribution for sulbactam-durlobactam and sulbactam against clinical isolates of ABC complex collected globally in 2016 and 2017

Category (no. of isolates), drug(s)a Frequency distribution (%) by indicated MIC (μg/ml)b
≤0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 >64
All ABC (1,722)
    SUL-DUR 0.2 0.3 2.8 13.0 42.0 71.8 90.9 97.7 98.6 98.8 99.4 99.9 100
    SUL 0 0 0 0.1 1.3 13.5 35.8 45.1 54.9 74.2 89.2 97.1 100
Asia/SP (221)
    SUL-DUR 0 0.5 2.3 9.0 44.3 78.7 94.6 98.2 98.2 98.6 99.1 100
    SUL 0 0 0 0 1.8 10.0 26.2 31.2 36.2 49.8 80.5 97.3 100
Europe (713)
    SUL-DUR 0.1 0.3 2.4 13.0 40.1 67.6 87.8 98.3 99.2 99.2 99.4 100
    SUL 0 0 0 0.1 1.3 13.3 37.0 44.0 52.0 71.0 87.5 97.2 100
Lat. Amer. (262)
    SUL-DUR 0 0 1.5 9.5 32.4 62.6 86.6 92.4 94.7 95.4 98.5 99.2 100
    SUL 0 0 0 0 0.4 6.9 19.8 26.0 36.3 72.9 85.5 92.0 100
Middle East (15)
    SUL-DUR 0 0 0 6.7 26.7 73.3 100
    SUL 0 0 0 0 0 0 6.7 20.0 66.7 93.3 100
N. Amer. (511)
    SUL-DUR 0.4 0.6 4.5 16.4 49.1 79.3 95.7 99.4 100
    SUL 0 0 0 0.2 1.6 19.0 47.4 63.2 76.1 89.4 97.1 99.4 100
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a

SUL-DUR, sulbactam-durlobactam; SUL, sulbactam; Asia/SP, Asia and South Pacific; Lat. Amer., Latin America; N. Amer., North America.

b

MIC90 is in boldface for each MIC distribution.

Against the 1,420 A. baumannii isolates tested, the SUL-DUR MIC50/MIC90 was 1/4 μg/ml (Table 1). SUL-DUR was slightly more active against the other Acinetobacter spp. tested, with a MIC50/MIC90 of 0.5/1 μg/ml against A. calcoaceticus and A. nosocomialis and a MIC50/MIC90 of 0.5/2 μg/ml against A. pittii. Generally, the A. baumannii isolates were less susceptible to the comparator agents, with only 37.0% and 35.6% susceptibility to imipenem and meropenem, respectively, compared to ≥93% susceptibility to carbapenems for the other Acinetobacter spp. tested.

The activity of SUL-DUR was stable across isolates from all the regions examined (Tables 1 and 2). SUL-DUR had a MIC50/MIC90 of 1/2 μg/ml against isolates from Asia and the South Pacific (n = 221), the Middle East (Israel only, n = 15), and North America (United States only, n = 551). Against isolates from Europe (n = 713) and Latin America (n = 262), SUL-DUR had a MIC50/MIC90 of 1/4 μg/ml. In comparison, susceptibility to imipenem ranged from 26.7% in the Middle East and Latin America to 65% in North America.

SUL-DUR activity was also consistent across isolates from different sources of infection (Table 1). SUL-DUR had a MIC50/MIC90 of 1/4 μg/ml against bloodstream isolates (n = 238). Against respiratory tract infection isolates (n = 1,056), urinary tract infection isolates (n = 316), intra-abdominal infection isolates (n = 65), and other or unknown sources of infection isolates (n = 33), the MIC50/MIC90 for SUL-DUR was 1/2 μg/ml. For isolates from skin and soft tissue infections (n = 14), the SUL-DUR MIC50/MIC90 was 0.5/1 μg/ml. In addition, SUL-DUR retained potency across drug-resistant ABC isolates (Table 3). The SUL-DUR MIC90 was 4 μg/ml for the following subsets: imipenem-nonsusceptible (n = 909), colistin-resistant (n = 81), minocycline-nonsusceptible (n = 337), ciprofloxacin-nonsusceptible (n = 1,017), and amikacin-nonsusceptible (n = 740) isolates. SUL-DUR also maintained a MIC90 of 4 μg/ml against multidrug resistant (MDR) (n = 259) and XDR (n = 32) isolates.

TABLE 3.

MIC cumulative frequency distribution for sulbactam-durlobactam and sulbactam against drug-resistant clinical isolates of ABC complex

Category (no. of isolates), drug(s)a Frequency distribution (%) by indicated MIC (μg/ml)b
≤0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 >64
All ABC (1,722)
    SUL-DUR 0.2 0.3 2.8 13.0 42.0 71.8 90.9 97.7 98.6 98.8 99.4 99.9 100
    SUL 0 0 0 0.1 1.3 13.5 35.8 45.1 54.9 74.2 89.2 97.1 100
IPM-NS (909)
    SUL-DUR 0 0 0.4 5.1 22.1 56.5 84.2 95.7 97.4 97.7 98.9 99.8 100
    SUL 0 0 0 0 0 0.1 1.0 5.2 19.3 53.2 80.1 94.6 100
CST-R (81)
    SUL-DUR 0 0 0 4.9 16.0 46.9 80.2 98.8 100
    SUL 0 0 0 0 0 2.5 6.2 12.3 33.3 61.7 82.7 97.5 100
MIN-NS (337)
    SUL-DUR 0 0 0 1.5 8.0 32.3 73.0 95.8 97.9 98.2 98.8 100
    SUL 0 0 0 0 0 0 0.6 3.6 12.8 39.2 76.9 94.1 100
CIP-NS (1,017)
    SUL-DUR 0 0 1.0 6.1 23.5 57.8 85.9 96.8 98.1 98.4 99.0 99.8 100
    SUL 0 0 0 0 0.1 1.5 4.9 11.6 26.0 57.7 82.8 95.8 100
AMK-NS (740)
    SUL-DUR 0 0 0.5 4.3 20.8 53.5 83.5 96.5 98.0 98.2 99.1 99.7 100
    SUL 0 0 0 0 0 0.1 0.8 5.4 18.1 48.9 78.6 94.7 100
MDR (259)
    SUL-DUR 0 0 0 1.2 6.2 30.1 69.5 95.0 97.3 97.7 98.5 100
    SUL 0 0 0 0 0 0 0 1.2 6.2 30.1 72.2 93.1 100
XDR (32)
    SUL-DUR 0 0 0 0 0 25.0 65.6 100
    SUL 0 0 0 0 0 0 0 0 3.1 25.0 65.6 93.8 100
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a

SUL-DUR, sulbactam-durlobactam; SUL, sulbactam; IPM-NS, imipenem nonsusceptible; CST-R, colistin resistant; MIN-NS, minocycline nonsusceptible; CIP-NS, ciprofloxacin nonsusceptible; AMK-NS, amikacin nonsusceptible; MDR, multidrug resistant (IPM-NS, MIN-NS, and AMK-NS); XDR, extensively drug resistant (IPM-NS, MIN-NS, AMK-NS, CIP-NS, CST-R, SUL-NS, and FEP-NS).

b

MIC90 is in boldface for each MIC distribution.

Characterization of SUL-DUR-resistant isolates.

Of the 1,722 ABC isolates tested, only 39 (2.3%) had SUL-DUR MIC values of >4 μg/ml, which is the proposed breakpoint based on extensive nonclinical and clinical pharmacokinetics/pharmacodynamics (PK/PD) analyses (15, 16). To understand the molecular drivers of SUL-DUR resistance, all 39 isolates with MIC values of >4 μg/ml were subjected to whole-genome sequencing. The whole-genome sequencing data were analyzed for multilocus sequence type (MLST), β-lactamase gene content, and variations in the efflux systems, outer membrane porin-like proteins, and PBPs. Table 4 summarizes the antibiotic susceptibilities, demographic information, and results from whole-genome sequencing of the SUL-DUR-resistant isolates. All 39 resistant isolates were A. baumannii, had SUL-DUR MIC values of 8 to >64 μg/ml, and were also resistant to imipenem. Only one of these isolates was resistant to colistin. Ten of the isolates were collected in 2016 and 29 in 2017. Analysis of the genome sequences revealed three different sets of genetically identical or clonal isolates from 2017. One set of clonal isolates was comprised of three isolates collected from the same hospital in Spain. Two additional, distinct sets of clonal isolates from the same hospital in Guatemala were also identified, one comprised of two isolates and another of six isolates. This may reflect either the predominance of certain strains in these two countries or a clonal outbreak(s) in these two hospitals. Only one isolate from each of these groups is shown in Table 4. Based on this analysis, there were 31 unique SUL-DUR-resistant isolates identified. The resistant isolates were collected in four different geographical regions: Asia (n = 4; Vietnam and Thailand), Europe (n = 10; Belgium, France, Spain, Greece, Italy, and Turkey), Latin America (n = 14; Mexico, Argentina, Colombia, Ecuador, and Guatemala), and North America (n = 3; United States).

TABLE 4.

Antibiotic susceptibility and demographic information for A. baumannii isolates with reduced susceptibility to sulbactam-durlobactam

Isolate Yr Country MIC (μg/ml) ofa:
 MLSTb (Oxford/Institut Pasteur) Efflux/porin variant(s)c,d bla variants PBP variant(s)d
SUL-DUR IPM AMK CIP CST MIN
1308777 2016 Vietnam 16 64 >64 >4 0.5 16 STOX1806, 208/STIP2 ND ADC-82, OXA-23, OXA-66 PBP3 (T526S)
1465642 2016 Belgium 64 64 4 >4 ≤0.25 ≤0.12 STOX1089/STIP85 ND OXA-94, NDM-1, subclass B3 MBL ND
1435703 2016 France 32 >64 2 >4 ≤0.25 0.5 STOX164/— ND CARB-2, OXA-58, OXA-91, NDM-1 ND
1407634 2016 Spain 8 >64 4 >4 ≤0.25 1 STOX106/STIP3 AdeI (E30K) ADC-7-like, OXA-24, OXA-71 PBP3 (T337I, G523V)
1407635 2016 Spain 8 64 >64 >4 0.5 16 STOX218/STIP2 ND ADC-30, OXA-23, OXA-66 ND
1420327 2016 Mexico 32 16 >64 >4 ≤0.25 8 STOX1806, 208/STIP2 ND ADC-30, TEM-1, OXA-83 PBP3 (A515V)
1386786 2016 Argentina 32 >64 >64 >4 0.5 1 STOX229/STIP25 ND ADC-26, OXA-64, PER-7, NDM-1 ND
1457337 2016 Argentina 16 32 16 >4 0.5 4 STOX1806, 208/STIP2 ND ADC-25, OXA-23, OXA-66 PBP3 (T526S)
1484127 2016 USA 8 64 >64 >4 0.5 8 STOX1806, 208/STIP2 ND ADC-30, OXA-23, OXA-66 PBP3 (T526S)
1483449 2016 USA 8 64 >64 >4 0.5 16 STOX1806, 208/STIP2 ND ADC-73, OXA-23, OXA-66 PBP3 (A515V)
1683261 2017 Thailand 64 >64 2 >4 ≤0.25 ≤0.12 STOX355/— No adeC ADC-169, CARB-2, OXA-402, NDM-1 ND
1683267 2017 Thailand 64 >64 >64 >4 1 8 STOX1838, 349/ST2IP ND ADC-73, TEM-1, OXA-23, OXA-66, NDM-1 ND
1662094 2017 Thailand 32 >64 32 >4 ≤0.25 1 STOX355/STIP16 No adeC ADC-169, OXA-58, OXA-402, VEB-1, NDM-1 ND
1589408 2017 Belgium 64 64 4 >4 ≤0.25 ≤0.12 STOX1089/STIP85 No adeC ADC-80 (V119E), ADC-176, OXA-94, NDM-1 ND
1620872 2017 Greece 8 64 >64 >4 1 8 STOX1816, 195/STIP2 adeA::Tn ADC-73, OXA-23, OXA-66 PBP1A (W343*), PBP3 (A515V)
1629634 2017 Italy 8 64 >64 >4 0.5 16 STOX1806, 208/STIP2 ND ADC-73, TEM-1, OXA-23, OXA-66 PBP3 (A515V)
1562926e 2017 Spain 64 64 64 >4 0.5 >16 STOX1489/STIP25 AdeJ (A290S), No adeC, adeS::Tn ADC-5 (G239S, N341T), OXA-23, OXA-64 PBP3 (T526S), PBP6b (T188S)
1557168 2017 Spain 8 64 2 >4 ≤0.25 16 STOX218/STIP2 ND OXA-23, OXA-66 PBP1A (W465*), PBP3 (K235N)
1557526 2017 Turkey 8 64 >64 >4 >8 2 STOX448/STIP2 AdeA (Q202L) ADC-30, TEM-1, OXA-23, OXA-66 PBP3 (F548I)
1625487 2017 Argentina 8 64 64 >4 1 2 STOX1806, 208/STIP2 ND ADC-25, OXA-23, OXA-66 PBP3 (T526S)
1647876 2017 Argentina 8 64 4 >4 ≤0.25 4 STOX1806, 208/STIP2 ND ADC-25, OXA-23, OXA-66 PBP3 (T526S)
1647764 2017 Argentina 8 64 64 >4 0.5 2 STOX1806, 208/STIP2 AdeH (Q79R) ADC-25, OXA-23, OXA-66 PBP3 (T526S)
1660516 2017 Colombia >64 >64 >64 >4 ≤0.25 4 —/STIP2 ND ADC-73, OXA-23, OXA-66, NDM-1 PBP3 (A515V)
1660477 2017 Colombia 8 >64 >64 >4 0.5 1 STOX124/STIP79 AdeC (Q93*) ADC-5, TEM-1: OXA-23, OXA-65 PBP1A (T117S)
1692917 2017 Ecuador 32 64 4 >4 ≤0.25 0.25 —/STIP126 No adeC ADC-50, OXA-58, OXA-64, NDM-1 ND
1699512f 2017 Guatemala 64 >64 >64 >4 0.5 4 —/STIP108 No adeC ADC-152 (S341T), TEM-1, OXA-132, NDM-1 PBP3 (V146I)
1699517g 2017 Guatemala 32 >64 2 0.25 0.5 ≤0.12 STOX1736/STIP734 AdeT (Q63P), CarO (W179fs) ADC-99-like, OXA-24, OXA-69 PBP2 (A133T), PBP3 (N377Y, T526S), PBP6b (A183V)
1699549 2017 Guatemala 16 64 >64 >4 0.5 2 —/STIP108 No adeC ADC-152 (S341T), OXA-24, OXA-132 PBP3 (Q488K)
1699548 2017 Guatemala 8 >64 >64 >4 0.5 1 —/STIP1 AdeC (P29L) ADC-53 (A236V), TEM-1, CTX-M-15, OXA-24, OXA-69 PBP3 (T526S)
1699513 2017 Guatemala >64 >64 64 >4 0.5 0.25 STOX514/STIP103 No adeC ADC-97-like, TEM-1, OXA-70, NDM-1 MtgA (F12I), PBP3 (N377Y, T526S)
1558650 2017 USA 8 32 >64 >4 ≤0.25 8 STOX1701/STIP2 ND ADC-25, OXA-23, OXA-66 PBP3 (F548I)
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a

SUL-DUR, sulbactam-durlobactam; IPM, imipenem; AMK, amikacin; CIP, ciprofloxacin; CST, colistin; MIN, minocycline.

b

MLST, multilocus sequence type determined by whole-genome sequencing; STOX/STIP, sequence type Oxford/Pasteur schemes.

c

ND, none detected.

d

*, stop codon.

e

Two additional clonal isolates identified.

f

One additional clonal isolate identified.

g

Five additional clonal isolates identified.

SUL-DUR-resistant isolates encoded either the blaNDM-1 metallo-β-lactamase or amino acid change(s) in PBP3 or both (Table 4). The 11 isolates that contained blaNDM-1 had SUL-DUR MIC values of 32 to >64 μg/ml. Durlobactam does not inhibit metallo-β-lactamases like NDM-1; therefore, it is not surprising that these isolates are nonsusceptible to SUL-DUR (6). There were 21 isolates found to encode amino acid changes in PBP3, which is the target of sulbactam inhibition. The most prevalent PBP3 mutant alleles were A515V (encoding a change of A to V at position 515) (n = 5) and T526S (n = 10). SUL-DUR MIC values for these isolates ranged from 8 μg/ml to >64 μg/ml; however, for the isolates with MIC values of >64 μg/ml, the blaNDM-1 gene was also present. Most of the isolates with A515V or T526S PBP3 mutations had MIC values of 8 to 32 μg/ml. Five other PBP3 mutants were also found in this surveillance study, but at much lower levels of prevalence; their mutations were T337I and G523V (n = 1), K235N (n = 1), F548I (n = 2), V146I (n = 1), and Q488K (n = 1). Two incidences of a truncated PBP1A were found, and some isolates encoded single amino acid changes in PBP2, MtgA, or PBP6b; however, most of these variants were only found in the presence of PBP3 changes, so it remains to be determined whether these variants in other cell wall synthesis proteins affect the activity of SUL-DUR.

The SUL-DUR-resistant isolates were also analyzed for mutations in efflux systems and outer membrane porins. Nine of the 31 unique SUL-DUR-resistant isolates lacked adeC, the outer membrane component of one of the RND efflux systems in Acinetobacter, which is found in about 20% of clinical isolates (17, 18). Several different single-amino-acid changes in components of the AdeABC, AdeFGH, and AdeIJK RND efflux systems were also observed. One isolate was found to encode a frameshift mutation in the CarO porin, which has been associated with carbapenem resistance (2). Additionally, an isolate with a transposon insertion in adeS, encoding part of the AdeRS two-component regulatory system for the regulation of the AdeABC efflux system, was found. Mutations in AdeRS can lead to overexpression of AdeABC and an increase in antibiotic efflux (19). For each of these mutations in efflux systems or porins, the isolate also encoded variations in the target of sulbactam (PBP3) and/or contained blaNDM-1, making it difficult to define the extent to which each of these changes affect SUL-DUR activity.

DISCUSSION

SUL-DUR demonstrated potent in vitro activity against clinical ABC isolates collected in 2016 and 2017 from around the globe. Of the isolates tested, 97.7% had a sulbactam-durlobactam MIC of ≤4 μg/ml, the proposed SUL-DUR breakpoint (15, 16). In contrast, only 45.1% of isolates had MIC values of ≤4 μg/ml for sulbactam alone, indicating that durlobactam effectively restores in vitro antibacterial activity to sulbactam. Susceptibility to SUL-DUR was higher than to all comparator agents tested against the ABC isolates in this study. The activity of SUL-DUR was consistent across geographical regions. This contrasts with activity observed for some of the comparator agents, such as imipenem, where susceptibility varied by region from 65% susceptible in North America to <35% susceptible in Latin America, Asia, and the Middle East. This variability in carbapenem resistance is consistent with other surveillance studies, which report higher rates of carbapenem resistance in Europe, Latin America, and some countries in Asia than in North America (2).

The activity of SUL-DUR was also stable across isolates from a variety of infection sources, including bloodstream and respiratory tract infections. Notably, SUL-DUR maintained potency against a variety of antibiotic-resistant subsets, including imipenem-nonsusceptible, colistin-resistant, minocycline-nonsusceptible, ciprofloxacin-nonsusceptible, amikacin-nonsusceptible, and MDR and XDR isolates.

A small percentage (2.3%) of isolates had SUL-DUR MIC values above the proposed breakpoint of 4 μg/ml. Among the SUL-DUR-nonsusceptible isolates, two previously identified mechanisms of resistance (6, 7, 20) were found to constitute the majority of resistant isolates found in this study. Most of the isolates encoded either the NDM-1 metallo β-lactamase, which is not inhibited by durlobactam, or amino acid change(s) in PBP3, the target of sulbactam (6, 7). Of note is that in this study, the prevalence of NDM-1 was less than 1% among all isolates, similar to what has been found in other ABC complex surveillance studies (6, 21). The most common PBP3 mutant alleles were the A515V and T526S variants. We have previously shown that spontaneous resistance to sulbactam alone and SUL-DUR in vitro maps to PBP3, resulting in mutants with reduced affinity for sulbactam, at low frequencies (7, 20). None of these PBP3 mutants isolated during spontaneous mutant selection in vitro were identified in this surveillance study; however, all of the amino acid substitutions in PBP3 identified in the current study are also located near the active-site serine (S336) (22), suggesting they may have reduced affinity for sulbactam.

Currently, because of antibiotic resistance, there are limited treatment options for infections caused by A. baumannii, resulting in >50% mortality (1, 2). A phase 3 study to evaluate the efficacy and safety of intravenous SUL-DUR in the treatment of patients with infections caused by ABC complex organisms is on-going (ClinicalTrials registration no. NCT03894046 [23]). The potent activity of SUL-DUR against recent, global clinical isolates of ABC organisms shown in this study suggests that SUL-DUR may be useful for the treatment of infections caused by A. baumannii, for which there is a great unmet medical need.

MATERIALS AND METHODS

Bacterial isolates.

A total of 1,722 ABC isolates were collected in 2016 and 2017 from 209 medical centers in 31 countries around the world. Each site was requested to collect clinical isolates from the ABC complex that were limited to one isolate per patient per year from hospitalized patients and to submit them to International Health Management Associates, Inc. (IHMA), in Schaumburg, IL, USA, for confirmatory identification and antimicrobial susceptibility testing. Due to the distribution of each of the ABC species in different geographical regions, this study did not evaluate the prevalence of each of these species in the regions examined. The identity of each isolate was confirmed using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) spectrometry (Bruker Daltonics, Billerica, MA, USA). The composition of ABC by species was as follows: A. baumannii, n = 1,420 (82.4%); A. calcoaceticus, n = 10 (0.6%); A. nosocomialis, n = 60 (3.5%); and A. pittii, n = 232 (13.5%).

The ABC isolates were collected from five geographical regions: Asia/South Pacific, n = 221 (12.8%) (Australia, Japan, Philippines, South Korea, Taiwan, Thailand, and Vietnam); Europe, n = 713 (41.4%) (Belgium, Czech Republic, France, Germany, Greece, Hungary, Italy, Portugal, Russia, Spain, Turkey, and United Kingdom); Latin America, n = 262 (15.2%) (Argentina, Brazil, Chile, Colombia, Ecuador, Guatemala, Mexico, Panama, Puerto Rico, and Venezuela); Middle East, n = 15 (0.9%) (Israel); and North America, n = 511 (29.7%) (United States). Isolates were taken from the following infection sources: bloodstream, n = 238 (13.9%); intra-abdominal, n = 65 (3.8%); respiratory tract, n = 1,056 (61.2%); urinary tract, n = 316 (18.3%); skin and soft tissue, n = 14 (0.8%); and other or unknown, n = 33 (2.0%).

Antimicrobial susceptibility testing.

Antimicrobial susceptibility testing was performed at IHMA using broth microdilution panels prepared in-house following standardized CLSI methods (24). Quality control testing was performed each day of testing as specified by CLSI using Escherichia coli strain ATCC 25922, Pseudomonas aeruginosa strain ATCC 27853, and A. baumannii strain NCTC 13304 (13). The SUL-DUR broth MIC quality control range is 0.5/4 to 2/4 μg/ml for A. baumannii NCTC 13304 (13). MIC values were interpreted using CLSI breakpoints for all antimicrobial agents except those for which CLSI breakpoints are not available (13). SUL-DUR was tested as 2-fold dilutions of sulbactam in combination with a fixed concentration of 4 μg/ml durlobactam. Isolates were categorized as MDR or XDR based on the criteria for Acinetobacter spp. outlined by Magiorakos et al. (25), which defines MDR as nonsusceptible to ≥1 agent in ≥3 classes and XDR as nonsusceptible to ≥1 agent in all but ≤2 classes of antimicrobials. The antimicrobial agents used for the MDR analysis were imipenem, minocycline, and amikacin. The antimicrobial agents used for the XDR analysis were imipenem, minocycline, amikacin, ciprofloxacin, colistin, cefepime, and sulbactam. For sulbactam, a susceptibility breakpoint of 4 μg/ml was used, which is based on the ampicillin-sulbactam (2:1) breakpoint of 8/4 μg/ml where sulbactam comprises the active component of the combination for Acinetobacter spp. (13).

Whole-genome sequencing and analysis.

Extraction of chromosomal DNA, whole-genome sequencing, and subsequent analysis of the genomic content of each isolate with a SUL-DUR MIC of >4 μg/ml was performed at Entasis Therapeutics. Chromosomal DNA was extracted from each isolate using the Promega Maxwell 16 instrument and Maxwell 16-cell DNA purification kit following the manufacturer’s protocol (Promega, Madison, WI). DNA was quantified with a Qubit 2.0 fluorometer using the double-stranded DNA (dsDNA) broad-range assay kit (Life Technologies, Grand Island, NY). DNA was diluted to 0.2 ng/μl, and a 5-μl amount was used for library generation using the Nextera XT DNA sample preparation kit and Nextera XT index primers (Illumina, San Diego, CA). The recommended procedure was followed, except that the library normalization step was omitted in favor of quantitative PCR (qPCR) library quantification. qPCR was performed on a Bio-Rad CFX96 cycler using the Kapa Biosystems library quantification kit (Kapa code KK4824) (Woburn, MA). Libraries were diluted to a standard concentration of 4 nM DNA, and 2.5 μl of each sample (8 to 12 samples, targeting 25- to 50-fold coverage) was combined and denatured with 0.1 N NaOH (final) for 5 min. The sample was diluted to 600 μl to provide a 15- to 20-pM multiplex library. Samples were sequenced on an Illumina MiSeq instrument using the V2 chemistry in a 2 × 150-bp paired-end read format.

Assembly and analysis of whole-genome sequencing was performed using CLC Genomics Workbench version 9.5 (CLCBio, Cambridge, MA). Fastq files were processed and analyzed as follows: duplicate sequence reads were removed, and remaining reads were trimmed for quality and minimum length (50 bp). Reads were de novo assembled at high stringency (fraction length = 0.9 and similarity fraction = 0.99) using default mismatch/insertion/deletion costs. Detection of single-nucleotide polymorphisms (SNPs) and indels was accomplished through mapping to a parent reference assembly using the same parameters. Quality-based SNPs were detected at a minimum frequency of 80% using default criteria.

Selected strains with reduced susceptibility to sulbactam-durlobactam were selected for sequence analysis of cell wall synthesis, efflux, and porin genes. The corresponding amino acid sequences were compared with the reference sequence of A. baumannii strain ATCC 17978 (GenBank accession number CP000521.1). The resultant variations in the amino acids of the proteins are listed in Table 4. The β-lactamase content of each strain was determined by BLAST within the CLC Genomics Workbench against an assembled database of genes curated at Entasis Therapeutics, with sequences originating from the NCBI Bacterial Antimicrobial Resistance Reference Gene Database (accession number PRJNA313047). For MLST determination, assembled contigs were exported from CLC Genomics Workbench and uploaded into the PubMLST database (https://pubmlst.org/databases/). For Acinetobacter, PubMLST hosts two different MLST schemes, Oxford and Institut Pasteur. The Oxford scheme (STox) assigns sequence types using the following genes: gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD. Alternatively, the Institut Pasteur scheme (STIP) assigns sequence types using alleles of cpn60, fusA, gltA, pyrG, recA, rplB, and rpoB. Sequence types from both schemes are reported when available.

ACKNOWLEDGMENTS

This study was sponsored by Entasis Therapeutics. M.A.H. is an employee of IHMA and had no personal financial interest in the sponsor of this paper (Entasis Therapeutics). S.M.M., S.H.M., and A.A.M. are employees and shareholders of Entasis Therapeutics.

All authors provided analysis input and have read and approved the final manuscript.

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