A novel antibiotic class targeting the lipopolysaccharide transporter

MIC determination

MIC determinations were performed using the broth microdilution method and in line with CLSI guidelines M07 and M100 (refs. 34,35). Bacterial inocula were prepared by diluting a 0.5 McFarland suspension in CAMHB or lysogeny broth (LB) specifically for A. baylyi isolates. Then, 96-well microtitre plates containing serial twofold dilution solutions of MCPs or standard of care antibiotics were inoculated with an appropriate volume of bacterial cells to give a final inoculum of around 5 × 105 CFU per ml and the desired test concentrations of antibacterial agents. The test plates were incubated for 20 to 24 h (for all Acinetobacter isolates) or 16 to 18 h (for non-Acinetobacter isolates) before visual inspection or reading the optical density at 600 nm (OD600) in the case of A. baylyi ATCC 33305 and the respective constructed mutants. MIC values of all of the tested antibiotics were read as the lowest compound concentration inhibiting bacterial growth by naked eye or beyond which the OD600 ceased to decrease. When testing MCP compounds using standard CAMHB non-supplemented medium, a trailing phenomenon was observed for part of the isolates rendering the MIC end-point reading ambiguous. Thus, for MCP tested in non-supplemented medium, the lowest concentration that demonstrated at least an 80% reduction in growth (MIC 80%) in comparison to the growth control was recorded, in addition to the MIC read as full growth inhibition (MIC 100%). Alternatively, the MIC testing was performed in CAMHB supplemented with 20–50% human serum, a condition that ameliorates trailing, and was read only at the end point of 100% growth inhibition.

Bacterial phenotypic fingerprint profiling

Sample preparation and analysis was conducted as previously described23 for A. baumannii, with the generated and analysed dataset consisting of three independent experiments, n = 3, as the only modification to the protocol.

Cell-viability assay

Compounds were prepared in serial dilutions (3.125–100 μM in 1% DMSO) and transferred to 384-well plates together with a positive (Staurosporine) and a negative (no compound) control. In total, 25 μl of HEK293 cells (ATCC; verified by short tandem repeat PCR and mycoplasma negative) (50,000 cells per ml) were added in either low-serum-containing (0.5% fetal bovine serum (FBS)) or high-serum-containing (12.5% FBS, 1% BSA) medium (Dulbecco’s modified Eagle medium (DMEM), glucose, l-glutamine). The plates were incubated for 24 h at 37 °C and a mixture of CellTiterGlo and DMEM was added to the plates. After incubation at room temperature, the luminescence signal was read using a BioTeK reader and a calculated IC50 was derived from the data.

Off-target activity screening

Off-target activity screening was conducted at Eurofins CEREP SA using the customized panel of 50 off-targets previously described36 and was run as single point measurements in duplicates in the presence of 10 μM of the test compound and reported as percentage inhibition of the radioligand signal or control enzymatic activity.

Rat plasma precipitation assay

Whole blood was obtained from WISTAR rats and collected in 1.2 ml heparin tubes for the preparation of heparin plasma. The tubes were then centrifuged for 5 min at 5,200g at room temperature to isolate the plasma supernatant. Test compounds were received as powder and solubilized at various concentrations in 0.9% aqueous sodium chloride solution and adjusted for pH with phosphate-buffered saline (PBS). The assay was conducted in 384-well plates. Rat plasma (10 µl) was added to 10 µl of the various compound solutions. A total of 10 µl of vehicle (0.9% aqueous sodium chloride solution, PBS) added to the plasma was used as a negative control. The absorption was measured at 362 nm. Raw data were obtained as absorbance data. The difference between the absorbance of the sample and the mean absorbance of the vehicle was calculated. The minimum effect concentration was defined as the lowest concentration giving an absorbance difference of OD362 ≥ 0.05.

Human plasma haemolysis and precipitation assay

For the in vitro haemolysis test, blood was provided by Roche medical services through an anonymous blood donation for research program, approved by the Ethics Committee Northwestern Switzerland and Central Switzerland (EKNZ) and collected with informed consent. Blood was collected by venipuncture in ethylenediaminetetraacetic acid (EDTA) and Li-heparin-coated tubes. Plasma was prepared from anticoagulated blood by centrifugation (2,500g; 10 min; at room temperature) and the haematocrit was measured to ensure that it was within normal reference values. Each formulation of compound in saline was added at a concentration of 8 mg ml−1 into test tubes containing exactly 0.5 ml of heparinated blood to give a final assay volume of 1 ml and a concentration of 4 mg ml−1 or less (following further dilutions). After incubation in a water bath for 10 min at 37 °C, the tubes were centrifuged (10 min, 1,811g at room temperature) and 100 μl of the supernatant was subsequently transferred into test tubes containing 5 ml Drabkins solution (RANDOX Laboratories) and 1 ml phosphate buffer. Haemoglobin was photometrically determined at 540 nm according to the RANDOX test-kit instructions. The results were expressed as percentage haemolysis extrapolated from an internal standard curve with 0%, 50% and 100% haemolysed blood samples.

For the plasma precipitation assay, test item formulations of compounds were diluted as follows with the vehicle (0.9% aqueous sodium chloride solution): undiluted, 1:2, 1:4, 1:8 and 1:16. These serial dilutions were then added into test tubes containing 0.5 ml of plasma to give a final assay volume of 1 ml with a final concentration of 4 mg ml−1 assay volume or less. After centrifugation of the samples (1,811g; 10 min; at room temperature), plasma precipitation was visually determined by scoring the resulting pellet (score: 0, none; 1, mild; 2, moderate; 3, marked).

Kinetic of killing determination

Time–kill studies were performed according to the CLSI standard procedure M26-A37. Zosurabalpin was tested at concentrations ranging from above, at and below MIC (twofold dilutions from 128 down to 0.25× MIC) and the control compound colistin was used at 4× MIC. A. baumannii colonies from agar plates were inoculated into CAMHB supplemented with 20% human serum and incubated overnight at 35 ± 2 °C in ambient air under shaking. The overnight culture was diluted 1:10,000 and further incubated for 2 h. A total of 5 ml of the bacterial log-phase suspension was transferred into six-well plates. A total of 50 μl of 100× antibiotic serial twofold dilution solutions was added according to the established multiple of MIC testing range. The six-well plates were incubated at 35 ± 2 °C in ambient air under shaking (100 rpm) for 24 h, 150 μl was withdrawn at the selected timepoints (0, 2, 4, 8, 12, 16, 20 and 24 h), diluted, plated on a Mueller–Hinton agar plate and incubated overnight for CFU determination. GraphPad Prism v.8 was used for graphical presentation of the data.

Resistance studies

For the single-step spontaneous mutation studies, four concentrations of zosurabalpin corresponding to multiples of agar MIC (2×, 4×, 8× and 16× MIC) were tested for each strain. Molten Mueller–Hinton agar (19 ml) supplemented with 20% human serum was added to 1 ml of compound at 20× the final concentration, poured immediately into Petri dishes (100 mm diameter) and gently mixed. Then, 2–3 colonies from agar plates were inoculated into CAMHB and incubated overnight under shaking (150 rpm). A total of 100 μl of the bacterial suspension (inoculum of around 108 CFU) was spread onto agar plates containing zosurabalpin and growth control plates without antibiotics. The agar plates were incubated aerobically at 35 ± 2 °C. After 24 h, the colonies grown on plates were counted and the spontaneous mutation frequencies were determined as the number of colonies counted on compound-plates divided by the inoculum size. Up to 8 colonies per condition including different morphology types were picked, tested for MIC determination and whole-genome sequenced.

Genomic characterization of mutants

Genomic DNA was extracted using the MagNA Pure Pathogen Universal Protocol 200 (MagNAPure 96 system, Roche) and used as the input for library preparation. For short-read sequencing, libraries were prepared using the Illumina Nextera XT library preparation kit (Illumina). The libraries were multiplexed, clustered and sequenced on the Illumina NextSeq system using a paired-end 150 bp cycles protocol at DDL Diagnostik Laboratory. Reads containing adapters and/or bacteriophage PhiX control sequences were removed and trimmed using Trimmomatic (v.0.36)38. Trimmed reads of parent strains were used to generate draft genomes by performing de novo assembly using SPAdes (v.3.12)39 with MismatchCorrector activated (–careful parameter) and annotation with Prokka (v.1.14.0)40 using the NCBI A. baumannii assembly (ASM975968v1; GCA_009759685.1) as the reference. Single-nucleotide polymorphism detection in derivative mutants was performed by mapping trimmed Illumina reads from derived colonies to the draft genome of the corresponding parent with the Genomic Short-read Nucleotide Alignment Program (GSNAP v2016-08-24)41 using the default parameters. Duplicate reads were removed using samtools42, awk scripts and Picard tools (Broad Institute). Variant calling was performed using Freebayes (v.1.1.0)43 followed by filtering using bcftools44 to remove variants present in the corresponding parent strain and requiring a read depth >5 and a variant frequency >0.8.

For long-read sequencing of genomic DNA, libraries were prepared using the amplification-free SQK-LSK109 library preparation protocol (Oxford Nanopore Technologies (ONT)). Libraries were multiplexed using either the EXP-NBD104 or EXP-NBD196 protocols (ONT), and sequenced on the ONT GridION Sequencer using an R9.4.1 flow cell over 72 h. Raw sequencing data were base-called and demultiplexed live during sequencing using Guppy (either v.3.2.8 or v.3.2.10) and a high-accuracy base-calling model (dna_r9.4.1_450bps_hac.cfg; ONT). Hybrid assemblies were generated first from raw ONT reads by CANU (v.2.0)45 followed by realigning ONT reads to the draft assembly by Minimap2 (v.2.17-r941)46. The alignment was used for assembly polishing first with ONT reads with Racon (v.1.4.16)47 and then by ten rounds of polishing using trimmed, unmapped Illumina reads by Pilon (v.1.23)48. Protein and gene annotations for polished hybrid assemblies were performed using Prokka (v.1.14.5)40 as described above. Hybrid assemblies were used to identify large insertions in derived mutants compared to parent strains using a sliding-window approach. Insertion elements were annotated using ISfinder (database from 10 November 2020)49.

Morbidostat-based experimental evolution and genomic profiling of zosurabalpin resistance

The experimental evolution approach using a custom-engineered continuous culturing device, morbidostat, was based on the principles previous introduced50. Implementation of morbidostat as well as the entire experimental and computational workflow were established and validated in model studies with triclosan in E. coli51 and ciprofloxacin in three Gram-negative species, including A. baumannii ATCC 17978 (ref. 25). In brief, the morbidostat-based workflow included: (1) competitive outgrowth of A. baumannii in 6 parallel reactors with regular computer-controlled medium dilutions leading to a gradual increase in drug concentration; (2) sequencing (with ~700–1,000× genomic coverage) of total genomic DNA from bacterial population samples taken as time series; (3) identification and quantitation of sequence variants (mutations, small insertion–deletion mutations, insertion sequences insertions, genomic rearrangements) to deduce evolutionary dynamics and resistance mechanisms; and (4) confirming the impact of major mutational variants by sequencing and MIC determination for selected clones. A. baumannii strains ATCC17978 and ATCC19606 were from ATCC, and two clinical isolates, ROB08705 and ROB08706 were from Roche collection. Starter cultures and growth media were as follows. Aliquots of glycerol stocks (from 6 colonies per strain) were grown to an OD600 of around 0.3 in CAMHB (TEKNOVA) at 37 °C and 2 ml of each culture was used to inoculate morbidostat reactors with 20 ml of the same medium with or without 20% human serum (Sigma-Aldrich, H4522). Drug dosing was as follows. A 10 mM stock solution of zosurabalpin compound in DMSO was used for preparing drug-containing medium and later for MIC measurements by serial dilutions in MHB medium with 2% DMSO in microtiter plates. Experimental evolution runs in morbidostat included two phases, starting with 2 μM zosurabalpin in drug medium until the intermediate resistance plateau was reached (typically within 48 h) followed by a tenfold increase in zosurabalpin up to 20 μM (over the next 24–36 h). Samples (10 ml) of evolving bacterial populations were typically taken once per day and were used to (1) isolate total genomic DNA for sequencing and (2) prepare glycerol stocks for further clonal analysis.

Genome sequencing and assembly for parental strains

All six starter cultures of each strain (A1–A6) were analysed using high-coverage Illumina sequencing (see below), assembly and RAST-based annotation as described for ATCC17978 strain25. The obtained genomic assemblies (provided in the Supplementary Information genome assembly file), including the identified pre-existing sequence variants, were used as a framework for the identification and analysis of sequence variants in evolved samples.

WGS of evolved population and isolate clones

DNA was extracted using the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich), analysed by Qbit and used for library preparation using one of the two protocols (kits): (1) the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England BioLabs) using TruSeq DNA UD Indexes 20022370 (IDT) without PCR amplification; or (2) the PlexWell PW384 kit with included adapters (seqWell). After quantification using quantitative PCR and quality-control analysis (2100 Bioanalyzer), the libraries were sequenced by Novogene (2 × 150 paired-end) with an average 500–1,000× genomic coverage for populations and 100–200× for isolated clones. To verify IS insertions, some of the clones were additionally analysed by Nanopore (MinION with FLO-MIN106 flow cell) sequencing using the Nanopore Rapid Barcoding kit SQK-RBK004 (Oxford Nanopore Technologies).

WGS data processing, variant calling and ranking

The computational pipeline for the initial variant calling was performed as described previously25 and is available for download online (https://docs.conda.io/projects/conda/en/latest/index.html). Potentially relevant non-pre-existing and non-synonymous mutational variants were ranked by (1) maximal relative abundance (Amax, %). All genes implicated by at least one event with Amax ≥ 10% distinct events with Amax ≥ 2% were selected for further ranking by (2) the number of independent occurrences (N) of the mutational events per gene (N ≥ 2).

SDS–PAGE and immunoblotting

Homemade Tris-HCl 4–20% polyacrylamide gradient gels or 4–20% Mini-PROTEAN TGX precast protein gels (Bio-Rad) were used with Tris-glycine running buffer. The 2× SDS sample loading buffer refers to a mixture containing 125 mM Tris (pH 6.8), 4% (w/v) SDS, 30% (v/v) glycerol, 0.005% bromophenol blue, and 5% (v/v) β-mercaptoethanol. SDS–PAGE gels were run for 45 to 60 min at 200 V. Protein complexes purified were analysed by SDS–PAGE followed by staining with Coomassie blue (Alfa Aesar) and imaging using the Gel feature of an Azure Biosystems C400 imager. For western blotting, proteins were transferred onto Immun-Blot PVDF membranes (Bio-Rad). Membranes were then blocked using sterile-filtered Casein blocking buffer (Sigma-Aldrich) for 1 h, and then incubated with the appropriate antibodies. Mouse monoclonal antiserum against the LPS core (Hycult Biotechnology), sheep anti-mouse horseradish peroxidase (HRP) conjugate secondary antibody (GE Amersham) and mouse anti-His tag HRP conjugate antibody (BioLegend) were used for the immunoblots. Bands were visualized using the ECL Prime Western blotting detection reagent (GE Amersham) and the Azure c400 imaging system. Uncropped immunoblots are provided in Supplementary Fig. 1.

Plasmids, strains and oligonucleotides

Genes encoding LptB, LptC and LptFG were amplified by PCR from A. baylyi ADP1 (ATCC 33305) genomic DNA. lptB and lptFG PCR products were inserted into pCDFduet by Gibson assembly (New England Biolabs) to generate plasmids analogous to those used for other LptB2FG homologues52. lptC PCR products were inserted into pET22/42 with a C-terminal thrombin cleavage site and a His7 tag. Oligonucleotide primers were purchased from Eton Biosciences or Genewiz. Plasmids and strains used in this study are reported in Supplementary Tables 8 and 9 with plasmid sequences provided.

Construction and use of mutant A. baylyi strains

Culture, genetic manipulation and MIC measurements of A. baylyi ADP1 were conducted according to previously reported procedures53,54. Point mutants were constructed in a two-step procedure as described previously55 with the introduction and excision of the integration cassette at codon 66 of pepA, wherein the excising fragment of otherwise wild-type chromosomal DNA sequence from codon 406 of pepA to codon 193 of lptG bore the desired mutation, and the resulting clones were screened by amplicon sequencing from codon 81 of holC to codon 501 of gpmI. After amplicon confirmation, three validated isolates of each constructed mutant were tested for susceptibility to a panel of antibiotics with known mechanisms of action as a further validation step to ensure congruence of phenotypes across replicates, which was confirmed in all cases, and one of the validated replicates was later used for MIC measurements reported here.

Purification of LptB2FGC complexes for biochemical reconstitution

LptB2FGC complexes were purified as previously described for LptB2FG with slight modifications56. Overnight cultures of E. coli C43(λDE3) containing pCDFduet-LptB-LptFG and pET22/42-LptC-thrombin-His7 were diluted 1:100 into LB containing 50 mg l−1 spectinomycin and 50 mg l−1 carbenicillin. Cells were grown at 37 °C to an OD600 of around 0.8. Then, 200 μM isopropyl β-d-1-thiogalactopyranoside (IPTG) and 0.2% glucose were added and cells were allowed to grow for another 2–3 h. Cells were collected by centrifugation (4,200g, 20 min, 4 °C). Cell pellets were flash-frozen using liquid nitrogen and stored at −80 °C. All of the subsequent steps were performed at 4 °C unless otherwise noted.

Thawed cell pellets were resuspended in lysis buffer (50 mM Tris (pH 7.4), 300 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 μg ml−1 lysozyme, 50 μg ml−1 DNase I, 1 cOmplete Protease Inhibitor Cocktail tablet per 40 ml) homogenized, and subjected to passage using an EmulsiFlex-C3 high-pressure cell disruptor three times. The cell lysate was centrifuged (10,000g, 10 min), and the supernatant was further centrifuged (100,000g, 1 h). The resulting pellets were resuspended and solubilized in solubilization buffer (20 mM Tris (pH 7.4), 300 mM NaCl, 15% glycerol, 5 mM MgCl2, 1% (w/v) DDM (Anatrace Maumee), 100 μM PMSF, 2 mM ATP) and rocked at 4 °C for 2 h. The mixture was centrifuged (100,000g, 30 min), and the supernatant was spiked with imidazole to a final concentration of 15 mM and then rocked with Ni-NTA Superflow resin (Qiagen) for 1 h. The resin was then washed with 2 × 10 column volumes affinity buffer (300 mM NaCl, 20 mM Tris (pH 7.4), 10% glycerol, 0.015% (w/v) DDM) containing 20 mM imidazole, followed by 2 × 15 column volumes affinity buffer containing 35 mM imidazole. Protein was eluted with 2 × 2 column volumes affinity buffer containing 200 mM imidazole, concentrated using a 100 kDa molecular mass cut-off Amicon Ultra centrifugal filter (Millipore) and purified by size-exclusion chromatography on a Superdex 200 increase column in SEC buffer (300 mM NaCl, 20 mM Tris (pH 7.4), 5% glycerol, 0.05% DDM, 0.5 mM tris(hydroxypropyl)phosphine). Fractions collected after size-exclusion chromatography were incubated overnight with restriction-grade thrombin (Sigma-Aldrich) to cleave the His tag. The solution was spiked with 8 mM imidazole, and the uncleaved protein was removed by passage through Ni-NTA resin and benzamidine Sepharose. The fractions were pooled, and concentrated to 7–8 mg ml−1 using a 100 kDa molecular mass cut-off Amicon Ultra centrifugal filter. Protein was then prepared in liposomes as described below.

Purification of LptAI36pBPA

LptAI36pBPA was purified as described previously56. In brief, E. coli BL21 (λDE3) cells containing pSup-BpaRS-6TRN and pET22b-LptA(I36Am) were grown to an OD600 of around 0.6 at 37 °C in LB medium containing 50 μg ml−1 carbenicillin, 30 μg ml−1 chloramphenicol and 0.8 mM ultraviolet-irradiation-cross-linkable amino acid p-benzoyl phenylalanine (pBPA) (BaChem). Cells were then induced with 50 μM isopropyl IPTG; allowed to grow for 2 h; collected; resuspended in a mixture containing 50 mM Tris-HCl (pH 7.4), 250 mM sucrose and 3 mM EDTA; incubated on ice for 30 min; and pelleted (6,000g, 10 min). The supernatant was supplemented with 1 mM PMSF and 10 mM imidazole and pelleted (100,000g, 30 min). The supernatant was incubated with Ni-NTA resin, which was then washed twice (20 column volumes of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% (v/v) glycerol and 20 mM imidazole). LptA was eluted twice (2.5 column volumes of wash buffer supplemented with an additional 180 mM imidazole), concentrated using a 10-kDa-cut-off Amicon centrifugal concentrator (Millipore), flash-frozen and stored at −80 °C until use.

Preparation of LptB2FGC liposomes

Proteoliposomes were prepared as described previously56. Aqueous E. coli polar lipid extract (Avanti Polar Lipids) (30 mg ml−1) and aqueous LPS from E. coli EH100 (Ra mutant; Sigma-Aldrich) (2 mg ml−1) were sonicated briefly for homogenization. A mixture of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 7.5 mg ml−1 E. coli polar lipids, 0.5 mg ml−1 LPS and 0.25% DDM was prepared and kept on ice for 10 min. Purified LptB2FGC was added to a final concentration of 0.86 μM, and the mixture was left on ice for 20 min. The mixture was diluted 100-fold with cold 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl and kept on ice for 20 min. The proteoliposomes were pelleted (300,000g, 2 h, 4 °C), resuspended in 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl, diluted 100× and centrifuged (300,000g, 2 h, 4 °C). The pellets were resuspended in a mixture of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10% glycerol (250 μl per 100 μl of the original predilution solution), homogenized by sonication, flash-frozen and stored at −80 °C until use.

LPS-release assay

The levels of release of LPS from proteoliposomes to LptA were measured as previously described52. Assays used 60% proteoliposomes (by volume) in a solution containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10% glycerol and 2 µM LptAI36pBPA. Reaction mixtures were incubated with drug for 10 min at room temperature, as applicable. Reactions were then initiated by the addition of ATP and MgCl2 (final concentrations of 5 mM and 2 mM, respectively) and proceeded at 30 °C. Aliquots (25 µl) were removed from the reaction mixtures and irradiated with ultraviolet light (365 nm) on ice for 10 min using a B-100AP lamp (Thermo Fisher Scientific). After ultraviolet irradiation, 25 µl 2× SDS–PAGE sample loading buffer was added, the samples were boiled for 10 min and proteins were separated using Tris-HCl 4–20% polyacrylamide gradient gels with Tris-glycine running buffer. Immunoblotting was conducted as described above.

Animal experiments ethical statement

Mouse pharmacokinetic studies and rat safety studies were conducted at Roche and all of the procedures were performed in accordance with the respective Swiss regulations and approved by the Cantonal Ethical Committee for Animal Research and conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) (animal research permit, 2395). The pharmacodynamics studies assessing the efficacy of the compounds were performed at Aptuit Verona, an Evotec company, and were subject to both the European directive 2010/63/UE governing animal welfare and protection, which is acknowledged by the Italian Legislative Decree no. 26/2014 and the company policy on the care and use of laboratory animals. All animals studies were revised by the Animal Welfare Body and approved by Italian Ministry of Health (51/2014-PR) and conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) (accredited unit, 001090). CD-1 mice were 6 weeks old at arrival (minimum acclimatization 5 days). Wistar Han IGS Crl:WI(Han) rats were 8 weeks old at the start of dosing. Mice were randomly allocated to treatment groups on arrival. Rats were randomly assigned to group/cage based on body weight.

Mouse pharmacokinetics study

Three male CD1 mice were administered with compound formulation (0.5 mg ml−1 in 0.9% aqueous sodium chloride solution) as an intravenous bolus dose of 1 mg per kg. Blood was sampled at 0.08, 0.25, 0.5, 1, 2, 4, 7 and 24 h after administration and the blood collecting tubes were centrifuged for 5 min at 5,200g at room temperature to isolate the plasma supernatant. The concentrations of compound in the plasma were analysed using a liquid chromatography–mass spectrometry method with a calibration range of 5–10,000 ng ml−1. The pharmacokinetic parameters were derived from the individual concentration data and were estimated by non-compartmental analysis.

Five-day repeat dose tolerability study

Four male Wistar rats per group were administered 0 (vehicle control), 0.6 or 6.0 mg per kg per day of RO7075573 or zosurabalpin as a slow intravenous infusion for five days (0, 0.12 or 1.2 mg ml−1 in 0.9% aqueous sodium chloride solution). Assessment of tolerability was based on mortality, in-life observations, body weight, food consumption and clinical pathology during the in-life phase. Moreover, gross pathology and histopathology were performed at unscheduled or scheduled euthanasia on day 6.

Immunocompetent mouse septicaemia infection model

Septicaemia was induced in CD-1 immunocompetent male mice by an intraperitoneal inoculation of a bacterial suspension of the tested A. baumannii isolate at a challenge of approximately 1–2 log[CFU] above the determined median lethal dose (resulting in 105 to 107 CFU per mouse). Doses of RO7075573 (ranging from 0.01 mg per kg to 1 mg per kg) or zosurabalpin (ranging from 0.3 to 30 mg per kg), of control standard-of-care antibiotic (meropenem tested at a single dose) and vehicle (sterile saline solution) were administered subcutaneously 1 and 5 h after infection. Mouse survival was followed over 6–7 days. GraphPad Prism 8 was used for graphical presentation of the data. Septicaemia studies with mutant derivative isolates obtained in resistance studies were performed as described above, using a bacterial challenge as determined for the parental isolate. Zosurabalpin was administered subcutaneously at a dose of 30 mg per kg twice.

Neutropaenic mouse thigh and lung infection model

Neutropenia was induced in male CD-1 mice by administration of two successive intraperitoneal injections on day −4 and day −1 of cyclophosphamide monohydrate (CPM) before the start of treatment with MCPs (RO7075573 or ZAB) or control standard of care antibiotics (colistin, meropenem or tigecycline). An intramuscular inoculation of a bacterial suspension of approximately 106 CFU per thigh was used to induce the infection. Treatment started 2 h after infection. Total doses of RO7075573, administered subcutaneously every 4 h, ranged from 1.8 to 180 mg per kg per day. Total doses of zosurabalpin, administered subcutaneously every 6 h, ranged from 6 to 360 mg per kg per day. Thigh bacterial burden was determined after 24 h of treatment. In the pneumonia model, an intratracheal inoculation of approximately 107 CFU per lung was used to induce the infection. Treatment started 2 h after infection. Total doses of zosurabalpin, administered subcutaneously every 6 h, ranged from 6 to 360 mg per kg per day. Lung bacterial burden was determined after 24 h treatment. Standard of care antibiotic was tested at a single dose in both infection models. GraphPad Prism 8 was used for graphical presentation and to analyse data.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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