Rational design of a new antibiotic class for drug-resistant infections.

The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including ß-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of ß-lactamases, the primary resistance mechanism associated with ß-lactam therapy in Gram-negative bacteria2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPE pathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years.

Characterization of Acinetobacter baumannii-calcoaceticus complex isolates and microbiological outcome for patients treated with sulbactam-durlobactam in a phase 3 trial (ATTACK).

Acinetobacter baumannii-calcoaceticus complex (ABC) causes severe, difficult-to-treat infections that are frequently antibiotic resistant. Sulbactam-durlobactam (SUL-DUR) is a targeted ß-lactam/ß-lactamase inhibitor combination antibiotic designed to treat ABC infections, including those caused by multidrug-resistant strains. In a global, pathogen-specific, randomized, controlled phase 3 trial (ATTACK), the efficacy and safety of SUL-DUR were compared to colistin, both dosed with imipenem-cilastatin as background therapy, in patients with serious infections caused by carbapenem-resistant ABC. Results from ATTACK showed that SUL-DUR met the criteria for non-inferiority to colistin for the primary efficacy endpoint of 28-day all-cause mortality with improved clinical and microbiological outcomes compared to colistin. This report describes the characterization of the baseline ABC isolates from patients enrolled in ATTACK, including an analysis of the correlation of microbiological outcomes with SUL-DUR MIC values and the molecular drivers of SUL-DUR resistance.

I n vitro pharmacokinetics/pharmacodynamics of the ß-lactamase inhibitor, durlobactam, in combination with sulbactam against Acinetobacter baumannii-calcoaceticus complex.

Infections caused by Acinetobacter baumannii are increasingly multidrug resistant and associated with high rates of morbidity and mortality. Sulbactam is a ß-lactamase inhibitor with intrinsic antibacterial activity against A. baumannii. Durlobactam is a non-ß-lactam ß-lactamase inhibitor with an extended spectrum of activity compared to other inhibitors of its class. In vitro pharmacodynamic infection models were undertaken to establish the pharmacokinetic/pharmacodynamic (PK/PD) index and magnitudes associated with sulbactam and durlobactam efficacy and to simulate epithelial lining fluid (ELF) exposures at clinical doses to understand sulbactam-durlobactam activity with and without co-administration of a carbapenem. Hollow fiber infection models (HFIMs) and one-compartment systems were used to identify the PK/PD indices and exposure magnitudes associated of 1-log10 and 2-log10 colony-forming unit (CFU)/mL reductions. Sulbactam and durlobactam demonstrated PK/PD drivers of % time above the minimum inhibition concentration (%T > MIC) and area under the plasma concentration-time curve from time 0 to 24 h (AUC0-24)/MIC, respectively. Against a sulbactam-susceptible strain, sulbactam %T > MIC of 71.5 and 82.0 were associated with 1-log10 and 2-log10 CFU/mL reductions, respectively, in the HFIM. Against a non-susceptible strain, durlobactam restored the activity of sulbactam with an AUC0-24/MICs of 34.0 and 46.8 using a polysulfone cartridge to achieve a 1-log10 and 2-log10 CFU/mL reduction. These magnitudes were reduced to 13.8 and 24.2, respectively, using a polyvinylidene fluoride cartridge with a membrane pore size of 0.1 µm. In the one-compartment model, durlobactam AUC0-24/MIC to achieve 1-log10 and 2-log10 CFU/mL reduction were 7.6 and 33.4, respectively. Simulations of clinical ELF exposures in the HFIM showed cidal activity at MICs ≤4 µg/mL. Penicillin binding protein 3 mutant strains with MICs of 8 µg/mL may benefit from the addition of a carbapenem at clinical exposures.

Sulbactam-durlobactam susceptibility test method development and quality control ranges for MIC and disk diffusion tests.

Sulbactam-durlobactam is a ß-lactam/ß-lactamase inhibitor combination developed to treat hospital-acquired and ventilator-associated bacterial pneumonia caused by Acinetobacter baumannii-calcoaceticus complex (ABC). Durlobactam is a diazabicyclooctane ß-lactamase inhibitor with potent activity against Ambler classes A, C, and D serine ß-lactamases and restores sulbactam activity against multidrug-resistant ABC. Studies were conducted to establish sulbactam-durlobactam antimicrobial susceptibility testing methods for both broth microdilution minimal inhibitory concentration (MIC) and disk diffusion tests as well as quality control (QC) ranges. To establish the MIC test method, combinations of sulbactam and durlobactam were evaluated using a panel of genetically characterized A. baumannii isolates which were categorized as predicted to be susceptible or resistant based on the spectrum of ß-lactamase inhibition by durlobactam. MIC testing with doubling dilutions of sulbactam with a fixed concentration of 4 µg/mL of durlobactam resulted in the greatest discrimination of the pre-defined susceptible and resistant strains. Similarly, the sulbactam/durlobactam 10/10 µg disk concentration showed the best discrimination as well as correlation with the MIC test. A. baumannii NCTC 13304 was selected for QC purposes because it assesses the activity of both sulbactam and durlobactam with clear endpoints. Multi-laboratory QC studies were conducted according to CLSI M23 Tier 2 criteria. A sulbactam-durlobactam broth MIC QC range of 0.5/4-2/4 µg/mL and a zone diameter QC range of 24-30 mm were determined for A. baumannii NCTC 13304 and have been approved by CLSI. These studies will enable clinical laboratories to perform susceptibility tests with accurate and reproducible methods.

Durlobactam, a Broad-Spectrum Serine ß-lactamase Inhibitor, Restores Sulbactam Activity Against Acinetobacter Species.

Sulbactam-durlobactam is a pathogen-targeted ß-lactam/ß-lactamase inhibitor combination in late-stage development for the treatment of Acinetobacter infections, including those caused by multidrug-resistant strains. Durlobactam is a member of the diazabicyclooctane class of ß-lactamase inhibitors with broad-spectrum serine ß-lactamase activity. Sulbactam is a first-generation, narrow-spectrum ß-lactamase inhibitor that also has intrinsic antibacterial activity against Acinetobacter spp. due to its ability to inhibit penicillin-binding proteins 1 and 3. The clinical utility of sulbactam for the treatment of contemporary Acinetobacter infections has been eroded over the last decades due to its susceptibility to cleavage by numerous ß-lactamases present in this species. However, when combined with durlobactam, the activity of sulbactam is restored against this problematic pathogen. The following summary describes what is known about the molecular drivers of activity and resistance as well as results from surveillance and in vivo efficacy studies for this novel combination.

Molecular drivers of resistance to sulbactam-durlobactam in contemporary clinical isolates of Acinetobacter baumannii.

Acinetobacter baumannii-calcoaceticus complex (ABC) causes severe infections that are difficult to treat due to pre-existing antibiotic resistance. Sulbactam-durlobactam (SUL-DUR) is a targeted ß-lactam/ß-lactamase inhibitor combination antibiotic designed to treat serious infections caused by Acinetobacter, including multidrug- and carbapenem-resistant strains. In a recent global surveillance study of 5,032 ABC clinical isolates collected from 2016 to 2021, less than 2% of ABC isolates had SUL-DUR MIC values >4 µg/mL. Molecular characterization of these isolates confirmed the primary drivers of resistance are metallo-ß-lactamases or penicillin-binding protein 3 (PBP3) mutations, as previously described. In addition, this study shows that certain common PBP3 variants, such as A515V, are insufficient to confer sulbactam resistance and that the efflux of durlobactam by AdeIJK is likely to play a role in a subset of strains.

In Vitro Activity of Sulbactam-Durlobactam against Global Isolates of Acinetobacter baumannii-calcoaceticus Complex Collected from 2016 to 2021.

Sulbactam-durlobactam is a ß-lactam-ß-lactamase inhibitor combination designed to treat serious Acinetobacter baumannii-calcoaceticus complex (ABC) infections, including carbapenem-non-susceptible and multidrug-resistant (MDR) isolates. The current study characterized the in vitro activity of sulbactam-durlobactam against a collection of 5,032 ABC clinical isolates collected in 33 countries across the Asia/South Pacific region, Europe, Latin America, the Middle East, and North America from 2016 to 2021. The sulbactam-durlobactam MIC50 and MIC90 were 1 and 2 µg/mL, respectively, for all ABC isolates tested. The addition of durlobactam (at a fixed concentration of 4 µg/mL) to sulbactam decreased its MIC50 by 8-fold (from 8 to 1 µg/mL) and its MIC90 by 32-fold (from 64 to 2 µg/mL) for all ABC isolates. The in vitro activity of sulbactam-durlobactam was maintained across individual ABC species, years, global regions of collection, specimen sources, and resistance phenotypes, including MDR and extensively drug-resistant (XDR) isolates. At 4 µg/mL (preliminary sulbactam-durlobactam susceptible MIC breakpoint), sulbactam-durlobactam inhibited 98.3% of all ABC isolates and >96% of sulbactam-, imipenem-, ciprofloxacin-, amikacin-, and minocycline-non-susceptible isolates; as well as colistin-resistant, MDR, and XDR isolates. Most imipenem-non-susceptible ABC isolates (96.8%, 2,488/2,570) were carbapenem-resistant A. baumannii (CRAB); 96.9% (2,410/2,488) of CRAB isolates were sulbactam-durlobactam-susceptible. More than 80% of ABC isolates had sulbactam-durlobactam MIC values that were ≥2 doubling-dilutions (4-fold) lower than sulbactam alone. Only 1.7% (84/5,032) of ABC isolates from 2016 to 2021 had sulbactam-durlobactam MIC values of >4 µg/mL. Of the 84 isolates, 94.0% were A. baumannii, 4.8% were A. pittii, and 1.2% were A. nosocomialis. In summary, sulbactam-durlobactam demonstrated potent antibacterial activity against a 2016 to 2021 collection of geographically diverse clinical isolates of ABC isolates, including carbapenem-non-susceptible and MDR isolates.

In Vitro Antibacterial Activity and In Vivo Efficacy of Sulbactam-Durlobactam against Pathogenic Burkholderia Species.

The Gram-negative bacterial genus Burkholderia includes several hard-to-treat human pathogens: two biothreat species, Burkholderia mallei (causing glanders) and B. pseudomallei (causing melioidosis), and the B. cepacia complex (BCC) and B. gladioli, which cause chronic lung infections in persons with cystic fibrosis. All Burkholderia spp. possess an Ambler class A Pen ß-lactamase, which confers resistance to ß-lactams. The ß-lactam-ß-lactamase inhibitor combination sulbactam-durlobactam (SUL-DUR) is in clinical development for the treatment of Acinetobacter infections. In this study, we evaluated SUL-DUR for in vitro and in vivo activity against Burkholderia clinical isolates. We measured MICs of SUL-DUR against BCC and B. gladioli (n = 150), B. mallei (n = 30), and B. pseudomallei (n = 28), studied the kinetics of inhibition of the PenA1 ß-lactamase from B. multivorans and the PenI ß-lactamase from B. pseudomallei by durlobactam, tested for blaPenA1 induction by SUL-DUR, and evaluated in vivo efficacy in a mouse model of melioidosis. SUL-DUR inhibited growth of 87.3% of the BCC and B. gladioli strains and 100% of the B. mallei and B. pseudomallei strains at 4/4 µg/ml. Durlobactam potently inhibited PenA1 and PenI with second-order rate constant for inactivation (k2/K) values of 3.9 × 106 M-1 s-1 and 2.6 × 103 M-1 s-1 and apparent Ki (Kiapp) of 15 nM and 241 nM, respectively, by forming highly stable covalent complexes. Neither sulbactam, durlobactam, nor SUL-DUR increased production of PenA1. SUL-DUR demonstrated activity in vivo in a murine melioidosis model. Taken together, these data suggest that SUL-DUR may be useful as a treatment for Burkholderia infections.

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

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 ß-lactam-ß-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-ß-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.

N-Hydroxyformamide LpxC inhibitors, their in vivo efficacy in a mouse Escherichia coli infection model, and their safety in a rat hemodynamic assay.

UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase (LpxC), the zinc metalloenzyme catalyzing the first committed step of lipid A biosynthesis in Gram-negative bacteria, has been a target for antibacterial drug discovery for many years. All inhibitor chemotypes reaching an advanced preclinical stage and clinical phase 1 have contained terminal hydroxamic acid, and none have been successfully advanced due, in part, to safety concerns, including hemodynamic effects. We hypothesized that the safety of LpxC inhibitors could be improved by replacing the terminal hydroxamic acid with a different zinc-binding group. After choosing an N-hydroxyformamide zinc-binding group, we investigated the structure-activity relationship of each part of the inhibitor scaffold with respect to Pseudomonas aeruginosa and Escherichia coli LpxC binding affinity, in vitro antibacterial potency and pharmacological properties. We identified a novel, potency-enhancing hydrophobic binding interaction for an LpxC inhibitor. We demonstrated in vivo efficacy of one compound in a neutropenic mouse E. coli infection model. Another compound was tested in a rat hemodynamic assay and was found to have a hypotensive effect. This result demonstrated that replacing the terminal hydroxamic acid with a different zinc-binding group was insufficient to avoid this previously recognized safety issue with LpxC inhibitors.

Determination of MIC Quality Control Ranges for the Novel Gyrase Inhibitor Zoliflodacin.

This report describes the results of two different, multilaboratory quality control (QC) studies that were used to establish QC ranges for the novel gyrase inhibitor zoliflodacin against the ATCC strains recommended by the Clinical and Laboratory Standards Institute (CLSI). Following the completion of an eight-laboratory, CLSI document M23-defined tier 2 study, the agar dilution MIC QC range for zoliflodacin against the Neisseria gonorrhoeae QC strain ATCC 49226 was defined as 0.06 to 0.5 µg/ml and was approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing. This QC range will be used for in vitro susceptibility testing of zoliflodacin during phase 3 human clinical trials and surveillance studies, and eventually it will be implemented in clinical labs. In a separate study, broth microdilution MIC quality control ranges for zoliflodacin against additional QC strains were determined to be 0.12 to 0.5 µg/ml for Staphylococcus aureus ATCC 29213, 0.25 to 2 µg/ml for Enterococcus faecalis ATCC 29212, 1 to 4 µg/ml for Escherichia coli ATCC 25922, 0.12 to 0.5 µg/ml for Streptococcus pneumoniae ATCC 49619, and 0.12 to 1 µg/ml for Haemophilus influenzae ATCC 49247. These MIC QC ranges were also approved by CLSI for use in future in vitro susceptibility testing studies against organisms other than N. gonorrhoeae.

Frequency and Mechanism of Spontaneous Resistance to Sulbactam Combined with the Novel ß-Lactamase Inhibitor ETX2514 in Clinical Isolates of Acinetobacter baumannii.

The novel diazabicyclooctenone ETX2514 is a potent, broad-spectrum serine ß-lactamase inhibitor that restores sulbactam activity against resistant Acinetobacter baumannii The frequency of spontaneous resistance to sulbactam-ETX2514 in clinical isolates was found to be 7.6 × 10-10 to <9.0 × 10-10 at 4× MIC and mapped to residues near the active site of penicillin binding protein 3 (PBP3). Purified mutant PBP3 proteins demonstrated reduced affinity for sulbactam. In a sulbactam-sensitive isolate, resistance also mapped to stringent response genes associated with resistance to PBP2 inhibitors, suggesting that in addition to ß-lactamase inhibition, ETX2514 may enhance sulbactam activity in A. baumannii via inhibition of PBP2.

Small-molecule inhibitors of gram-negative lipoprotein trafficking discovered by phenotypic screening.

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.

Novel antibacterial targets and compounds revealed by a high-throughput cell wall reporter assay.

UNLABELLED: A high-throughput phenotypic screen based on a Citrobacter freundii AmpC reporter expressed in Escherichia coli was executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action. E. coli mutants resistant to these compounds display no cross-resistance to antibiotics of other classes. Resistance to the sulfonyl piperazine maps to LpxH, which catalyzes the fourth step in the synthesis of lipid A, the outer membrane anchor of lipopolysaccharide (LPS). To our knowledge, this compound is the first reported inhibitor of LpxH. Resistance to the pyrazole compound mapped to mutations in either LolC or LolE, components of the essential LolCDE transporter complex, which is required for trafficking of lipoproteins to the outer membrane. Biochemical experiments with E. coli spheroplasts showed that the pyrazole compound is capable of inhibiting the release of lipoproteins from the inner membrane. Both of these compounds have significant promise as chemical probes to further interrogate the potential of these novel cell wall components for antimicrobial therapy. IMPORTANCE: The prevalence of antibacterial resistance, particularly among Gram-negative organisms, signals a need for novel antibacterial agents. A phenotypic screen using AmpC as a sensor for compounds that inhibit processes involved in Gram-negative envelope biogenesis led to the identification of two novel inhibitors with unique mechanisms of action targeting Escherichia coli outer membrane biogenesis. One compound inhibits the transport system for lipoprotein transport to the outer membrane, while the other compound inhibits synthesis of lipopolysaccharide. These results indicate that it is still possible to uncover new compounds with intrinsic antibacterial activity that inhibit novel targets related to the cell envelope, suggesting that the Gram-negative cell envelope still has untapped potential for therapeutic intervention.

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

Sulbactam is a class A ß-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.

Pharmacodynamic Profiling of a Siderophore-Conjugated Monocarbam in Pseudomonas aeruginosa: Assessing the Risk for Resistance and Attenuated Efficacy.

The objective of this study was to investigate the risk of attenuated efficacy due to adaptive resistance for the siderophore-conjugated monocarbam SMC-3176 in Pseudomonas aeruginosa by using a pharmacokinetic/pharmacodynamic (PK/PD) approach. MICs were determined in cation-adjusted Mueller-Hinton broth (MHB) and in Chelex-treated, dialyzed MHB (CDMHB). Spontaneous resistance was assessed at 2× to 16× the MIC and the resulting mutants sequenced. Efficacy was evaluated in a neutropenic mouse thigh model at 3.13 to 400 mg/kg of body weight every 3 h for 24 h and analyzed for association with free time above the MIC (fT>MIC). To closer emulate the conditions of the in vivo model, we developed a novel assay testing activity mouse whole blood (WB). All mutations were found in genes related to iron uptake: piuA, piuC, pirR, fecI, and pvdS. Against four P. aeruginosa isolates, SMC-3176 displayed predictable efficacy corresponding to the fT>MIC using the MIC in CDMHB (R(2) = 0.968 to 0.985), with stasis to 2-log kill achieved at 59.4 to 81.1%. Efficacy did not translate for P. aeruginosa isolate JJ 4-36, as the in vivo responses were inconsistent with fT>MIC exposures and implied a threshold concentration that was greater than the MIC. The results of the mouse WB assay indicated that efficacy was not predictable using the MIC for JJ 4-36 and four additional isolates, against which in vivo failures of another siderophore-conjugated ß-lactam were previously reported. SMC-3176 carries a risk of attenuated efficacy in P. aeruginosa due to rapid adaptive resistance preventing entry via the siderophore-mediated iron uptake systems. Substantial in vivo testing is warranted for compounds using the siderophore approach to thoroughly screen for this in vitro-in vivo disconnect in P. aeruginosa.

Discovery and Characterization of New Hydroxamate Siderophores, Baumannoferrin A and B, produced by Acinetobacter baumannii.

Acinetobacter baumannii AYE does not produce acinetobactin but grows under iron limitation. Accordingly, analyses of AYE iron-restricted culture supernatants resulted in the isolation of two fractions, which contained only hydroxamates and showed siderophore activity. Structural analyses identified baumannoferrin A and baumannoferrin B, which differ only by a double bond. These siderophores are composed of citrate, 1,3-diaminopropane, 2,4-diaminobutyrate, decenoic acid, and α-ketoglutarate. Analysis of the AYE genome showed the presence of a 12-gene cluster coding for proteins similar to those involved in the production and utilization of the hydroxamate siderophores acinetoferrin and achromobactin. As A. baumannii AYE does not produce acinetobactin and harbors only one gene cluster encoding the production and utilization of a siderophore, this strain's growth under iron limitation depends on baumannoferrin, a novel hydroxamate that could play a role in its virulence.

In vitro antibacterial activity of sulbactam-durlobactam in combination with other antimicrobial agents against Acinetobacter baumannii-calcoaceticus complex.

Combinations of the ß-lactam/ß-lactamase inhibitor sulbactam-durlobactam and seventeen antimicrobial agents were tested against strains of Acinetobacter baumannii in checkerboard assays. Most combinations resulted in indifference with no instances of antagonism. These results suggest sulbactam-durlobactam antibacterial activity against A. baumannii is unlikely to be affected if co-dosed with other antimicrobial agents.

Clinical Outcomes for Patients With Monomicrobial vs Polymicrobial Acinetobacter baumannii-calcoaceticus Complex Infections Treated With Sulbactam-Durlobactam or Colistin: A Subset Analysis From a Phase 3 Clinical Trial.

Background: In a previous study, the efficacy and safety of sulbactam-durlobactam vs colistin for the treatment of patients with carbapenem-resistant Acinetobacter baumannii-calcoaceticus complex (CRABC) infections were evaluated in a randomized controlled phase 3 trial. Both arms were dosed on a background of imipenem-cilastatin to treat coinfecting gram-negative pathogens. Thirty-six percent of infections in the primary efficacy population were polymicrobial. Methods: A subset analysis was performed to compare clinical and microbiological outcomes at test of cure (7 ± 2 days after the last dose) for patients with monomicrobial and polymicrobial CRABC infections. Minimal inhibitory concentrations of antibiotics against baseline isolates were determined by broth microdilution according to Clinical and Laboratory Standards Institute methodology. Results: Clinical cure, 28-day all-cause mortality, and microbiological outcomes were similar for patients in the sulbactam-durlobactam treatment arm with monomicrobial or polymicrobial A baumannii-calcoaceticus infections. Patients in the colistin arm with monomicrobial CRABC infections had higher mortality rates with worse clinical and microbiological outcomes as compared with those with polymicrobial infections. For patients who received sulbactam-durlobactam, imipenem susceptibility of coinfecting gram-negative pathogens trended with clinical benefit for patients with polymicrobial A baumannii-calcoaceticus infections. When tested in vitro, durlobactam restored imipenem susceptibility to the majority of coinfecting gram-negative pathogens from the sulbactam-durlobactam arm. This phenotype appeared to be related to the clinical outcome in 13 of 15 evaluable cases. Conclusions: These results suggest that the use of sulbactam-durlobactam plus a carbapenem could be an effective approach to treat polymicrobial infections that include CRABC, but additional clinical data are needed to demonstrate efficacy.

Inhibiting Mycobacterium abscessus Cell Wall Synthesis: Using a Novel Diazabicyclooctane ß-Lactamase Inhibitor To Augment ß-Lactam Action.

Mycobacterium abscessus (Mab) infections are a growing menace to the health of many patients, especially those suffering from structural lung disease and cystic fibrosis. With multidrug resistance a common feature and a growing understanding of peptidoglycan synthesis in Mab, it is advantageous to identify potent ß-lactam and ß-lactamase inhibitor combinations that can effectively disrupt cell wall synthesis. To improve existing therapeutic regimens to address serious Mab infections, we evaluated the ability of durlobactam (DUR), a novel diazobicyclooctane ß-lactamase inhibitor to restore in vitro susceptibilities in combination with ß-lactams and provide a biochemical rationale for the activity of this compound. In cell-based assays, susceptibility of Mab subsp. abscessus isolates to amoxicillin (AMOX), imipenem (IMI), and cefuroxime (CXM) was significantly enhanced with the addition of DUR. The triple drug combinations of CXM-DUR-AMOX and IMI-DUR-AMOX were most potent, with MIC ranges of ≤0.06 to 1 µg/mL and an MIC50/MIC90 of ≤0.06/0.25 µg/mL, respectively. We propose a model by which this enhancement may occur, DUR potently inhibited the ß-lactamase BlaMab with a relative Michaelis constant (Ki app) of 4 × 10-3 ± 0.8 × 10-3 µM and acylation rate (k2/K) of 1 × 107 M-1 s-1. Timed mass spectrometry captured stable formation of carbamoyl-enzyme complexes between DUR and LdtMab2-4 and Mab d,d-carboxypeptidase, potentially contributing to the intrinsic activity of DUR. Molecular modeling showed unique and favorable interactions of DUR as a BlaMab inhibitor. Similarly, modeling showed how DUR might form stable Michaelis-Menten complexes with LdtMab2-4 and Mab d,d-carboxypeptidase. The ability of DUR combined with amoxicillin or cefuroxime and imipenem to inactivate multiple targets such as d,d-carboxypeptidase and LdtMab2,4 supports new therapeutic approaches using ß-lactams in eradicating Mab. IMPORTANCE Durlobactam (DUR) is a potent inhibitor of BlaMab and provides protection of amoxicillin and imipenem against hydrolysis. DUR has intrinsic activity and forms stable acyl-enzyme complexes with LdtMab2 and LdtMab4. The ability of DUR to protect amoxicillin and imipenem against BlaMab and its intrinsic activity along with the dual ß-lactam target redundancy can explain the rationale behind the potent activity of this combination.