Bacterial strains and antibiotics
The P. aeruginosa PAO1 reference strain and five isogenic strains producing the MBLs NDM-1, IMP-1, IMP-10, IMP-13, and VIM-2 were used. The MBL genes blaNDM-1, blaIMP-1, blaIMP-10, blaIMP-13, and blaVIM-2 were amplified using the Platinum™ SuperFi II PCR mix (ThermoFisher, Madrid, Spain) and the primers listed in Table 3. The amplicons were then ligated into a high-copy-number vector, pUCP24, and transformed into E. coli Top10. Transformants were selected on Luria–Bertani (LB, Sigma-Aldrich, Madrid, Spain) agar plates supplemented with 15 mg/L gentamicin and confirmed using PCR and sequencing of the MBL allele. Recombinant plasmids were then transformed in PAO1 using electroporation27.
Imipenem was used as a standard laboratory powder from Sigma (Sigma-Aldrich, Madrid, Spain) for in vitro studies, while clinical formulations (Fresenius Kabi, Barcelona, Spain) were used for in vivo experiments. DMSA was used as a standard laboratory powder (Sigma-Aldrich, Madrid, Spain) for both in vitro and in vivo studies.
In vitro studies
In vitro studies were conducted in triplicate on three different days to avoid possible bias.
Antimicrobial susceptibility testing
The MICs of imipenem and DMSA were determined using the broth microdilution method with two-fold serial dilutions of both agents (ranging from 2 to 1024 mg/L), following the EUCAST guidelines (EUCAST, 2025)28. Tests were conducted using Cation-adjusted Müller–Hinton Broth II (CAMHBII, Merck Life Science S.L., Madrid, Spain) and a starting inoculum of 5 × 105 CFU/mL. P. aeruginosa ATCC 27853 was used as a quality control strain. The MIC was defined as the lowest concentration of antibiotic at which no growth was visible, evaluated after 20–24 h of incubation at 37 °C. The imipenem results were interpreted according to the EUCAST breakpoints28. Moreover, the MIC of imipenem in the presence of DMSA at a fixed concentration of 3 mM was also determined using the broth microdilution method, according to EUCAST28.
Time–kill assays
Imipenem was assayed at MIC values alone and in combination with DMSA at 3 mM for all six strains. The synergy studies were carried out using CAMHBII (Merck Life Science S.L., Madrid, Spain) with a starting inoculum of 5 × 105 CFU/mL and the drugs alone or in combination. The tubes were incubated at 37 °C with shaking, and samples were taken at 0, 2, 4, 8, and 24 h, serially diluted, and seeded in 5% sheep blood plates29. Bactericidal activity was defined as a decrease of ≥ 3 log10 CFU/mL from the initial inoculum. Synergistic activity was defined as a decrease of ≥ 2 log10 CFU/mL for the antimicrobial combination compared with the most active single agent30.
In vivo studies
Ethics and animals
Animal studies were carried out following the recommendations in the Guide for the Care and Use of Laboratory Animals31 and after obtaining the approval of the Committee on the Ethics of Animal Experiments of the Hospitales Universitarios Virgen Macarena y Virgen del Rocío (Exp.Animal_1703-N-23) and the Consejería De Agricultura, Pesca, Agua y Desarrollo Rural (27/11/2024/166), Junta de Andalucia, Spain. This study complies with ARRIVE guidelines (https://arriveguidelines.org/). Immunocompetent CD-1 female mice were used weighing 30 g (7–9 weeks old) (Production and Experimentation Animal Centre, Janvier Labs, Le Genest-Saint-Isle, France). The mice were housed in a ventilated cage system under specific pathogen-free conditions, with water and food ad libitum.
Toxicity studies
In a set of five female mice, the imipenem dosing schedule (100 mg/kg/ip/q2h/24h) was evaluated prior to efficacy studies. The following signs of acute (single-dose) and cumulative (full therapeutic treatment of the efficacy model) toxicity were assessed for 7 days: reduced water intake (dehydration)/food intake, isolation, self-mutilation, tremors/spasms, dyspnea, physical activity (increased/reduced), chromo-dacryorrhea, dermal responses (erythema/edema/redness/discoloration/necrosis), muscle stiffness, piloerection, teeth grinding, and weight loss. Toxicity studies of DMSA previously reported an LD of 2500 mg/kg/ip, with no toxicity for the selected dose (200 mg/kg/ip)17.
Pharmacokinetic/pharmacodynamic analysis
Imipenem serum concentrations were determined in 30 healthy CD-1 female mice after a single dose of imipenem (100 mg/kg/ip). Briefly, blood samples were obtained from groups of randomly anesthetized mice (N = 3) from the periorbital plexus at the following time-points after imipenem administration: 0, 5, 10, 15, 30, 60, 120, 240, 480, and 1440 min. The collected blood samples were centrifuged (4500 rpm for 15 min at 4 °C) and stored at − 80 °C until analysis. Both the serum-free and total antibiotic concentrations were measured using high-performance liquid chromatography (HPLC)–tandem mass spectrometry (LC-MS/MS) as previously described32. The imipenem maximum concentration of drug in the serum (Cmax), elimination half-life (t1/2), and area under the concentration–time curve from 0 to 24 h (AUC0−24) were calculated using PKSolver33. The pharmacodynamic (PD) target to evaluate the efficacy of imipenem was the serum concentration time above the MIC (%T>MIC). The dose regime of DMSA was based on previously reported pharmacokinetics data, in which the serum Cmax in healthy mice was 2.02 mg/L (range 1.59–3.36)17.
Peritoneal sepsis model
To evaluate the in vivo efficacy of imipenem alone and in combination with DMSA, a severe experimental peritoneal sepsis model with secondary BSI was used34. Firstly, the MLD was calculated for each of the six P. aeruginosa strains selected in the study, defined as the concentration that kills 100% of the animals inoculated35. Briefly, groups of six unanesthetized mice were ip-inoculated with 0.5 mL of increasing bacterial concentrations, starting from an inoculum of 6 log10 CFU/mL and proceeding until the MLD was achieved for each strain. After inoculation, the animals were observed hourly for the first 12 h and then daily for 7 days.
After characterization of the MLD for each isolate, we evaluated the efficacy of imipenem alone and in combination with DMSA. Briefly, for each strain, groups of mice (N = 5–10) were ip-inoculated with 0.5 mL of their MLD (log10 CFU/mL). Therapy began 2 h after inoculation and lasted for 24 h. The infected mice were randomly assigned to the following groups: (i) control (infected and untreated); (ii) imipenem 100 mg/kg/q2h/ip; (iii) DMSA 200 mg/kg/q4h/ip; or (iv) imipenem plus DMSA. Samples were aseptically extracted and processed immediately after the death of the mouse or after sacrifice at the end of the study (sodium thiopental/ip). Through cardiac punctures, blood samples were obtained for quantitative (log10 CFU/mL) and qualitative blood cultures (expressed as positive or negative). Spleens were aseptically extracted, weighed, and homogenized in sterile saline (Stomacher 80; Tekmar Co., Cincinnati, OH, USA) before quantitative cultures (log10 CFU/g) in Columbia agar with 5% sheep blood plates.
Statistical analysis
Mortality and positive blood cultures are expressed as percentages, while bacterial spleen concentrations (log10 CFU/g) and bacterial blood concentrations (log10 CFU/mL) are expressed as means ± SDs. Differences in bacterial concentrations were compared using Mann–Whitney U tests. Mortality and blood sterility rates between groups were compared using a two-tailed Fisher’s test. A p-value < 0.05 was considered significant, and SPSS v26.0 was used (SPSS Inc., Chicago, IL, USA).
