A novel antibiotic class targeting the enolase of Acinetobacter baumannii

A novel antibiotic class targeting the enolase of Acinetobacter baumannii | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A novel antibiotic class targeting the enolase of Acinetobacter baumannii Younes Smani, Irene Molina Panadero, Antonio Moreno Rodríguez, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5059044/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract High-throughput screening studies provide an additional approach to discovering repurposed drugs for antimicrobial treatments. In this work, we report the identification of ENOblock, an anticancer drug, as a novel antibiotic class. We computationally and experimentally validated that ENOblock synergizes with the last resort antibiotic, the colistin. Additionally, we identified enolase as the potential bacterial target for ENOblock. The in silico and in vitro antibacterial activity of ENOblock translated into potent in vivo efficacy in animal infection models. Collectively, the preclinical data support the selection of ENOblock as a promising candidate for antimicrobial development, with the potential to address the urgent threat of infections caused by Acinetobacter baumannii . Biological sciences/Microbiology/Antimicrobials Health sciences/Pathogenesis/Infection ENOblock enolase treatment bacteria infection Acinetobacter baumannii Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Gram-negative bacteria (GNB) are a significant concern in healthcare settings due to their ability to cause a wide range of infections such as pneumonia, bloodstream infections, wound or surgical site infections, and meningitis 1 , that are often difficult to treat. GNB infections, especially hospital acquired ones, pose a significant burden on healthcare systems worldwide, with reported costs of $ 136 million per year 2 , requiring ongoing efforts in surveillance, infection prevention and control, antibiotic stewardship, and research into new treatment options to mitigate their impact. One of the main difficulties in tackling GNB is their high efficiency in acquiring antimicrobial resistance (AMR) encoded by genomic, transcriptomic and proteomic changes 3 . Compounding the problem of AMR with reported high number of deaths associated with bacterial AMR and/or attributable to bacterial AMR is the immediate threat of a reduction in the discovery and development of new antibiotics. The World Health Organization (WHO) 4 and other European institutions have recently underscored the dangers posed by these infections 5 . Consequently, a perfect storm is converging regarding these infections: increasing antimicrobial resistance with a decreased new drug development 5 . This context is likely the best example of the purported “Post-Antibiotic Era”, with relevance even in non-specialized media. It is clear that new policies and actions are necessary to avoid the forecasts for 2050 that attribute ten million deaths worldwide to antimicrobial resistance 5 . Especially for pathogens like Acinetobacter baumannii that is reported with continuous increase in AMR and are accounted for most reported associated diseases and deaths 6 . Approaches using computational methods and high-throughput screening (HTS) have recently been developed for antibiotic discovery 7 – 10 . For example, screening small-molecule libraries has revealed new antimicrobial agents that belong to existing or new antibiotic classes 11 – 13 . Recently, HTS studies have been developed to discover repurposed drugs for antimicrobial treatments 14 . Previously, we identified anticancer drugs such as Selective Estrogen Receptor Modulators - specifically tamoxifen and its metabolites - as new agents for the treatment of A. baumannii 15 – 17 . In this work, we report the identification of ENOblock, an anticancer drug, as a novel antibiotic class. We computationally and experimentally validated that ENOblock synergizes with the last resort antibiotic, the colistin. Additionally, we identified enolase as the potential bacterial target for ENOblock. The in silico and in vitro antibacterial activity of ENOblock translated into potent in vivo efficacy in animal models of infection. Collectively, these preclinical data could support the selection of ENOblock as a promising candidate for antimicrobial development with the potential to address the urgent threat of infections caused by A. baumannii . MATERIAL AND METHODS Bacterial Strains A total of 32 clinical strains of A. baumannii colistin-resistant (n = 14) 18,19 or carbapenem-intermediate/resistant (n = 18) were collected from the "II Spanish Study of A. baumannii GEIH-REIPI 2000–2010" multicenter study (GenBank Bioproject PRJNA422585) and the reference strain ATCC 17978 were used in this study. HTS was performed with two reference strains of A. baumannii and Escherichia coli (ATCC 17978 and ATCC 25922, respectively) and four well-characterized, clonally unrelated clinical strains: A. baumannii Ab9 (ST672), colistin- and tigecycline-susceptible; MDR A. baumannii Ab186 (ST208), colistin-susceptible, tigecycline-resistant; E. coli C1-7-LE (ST8671), colistin- and tigecycline-susceptible; and MDR E. coli MCR1 + (ST6108), colistin-resistant, tigecycline-susceptible 20 . EU-OPENSCREEN library and HTS validation EU-OPENSCREEN provided a subset of 2,464 bioactive compounds from the ECBL pilot library (EU-OPENSCREEN: European Chemical Biology Library - Pilot Library) as 10 mM stock solutions in 100% DMSO. The library was screened in duplicate at a final concentration of 100 µM per well (1% DMSO). The antibacterial single-concentration HTS and dose-response susceptibility assays were conducted in 384-well plates, with bacterial cell density measured by optical density at 600 nm (OD600). A starting inoculum of 10 6 colony-forming units (CFU)/mL was used, with an incubation time of 24 hours for the A. baumannii (ATCC 17978, Ab9, and Ab186) strains and E. coli (ATCC 25922, C17LE, and MCR1 + ) strains. Imipenem and colistin were used as internal controls for A. baumannii and E. coli strains, respectively. Compounds were distributed in duplicate on separate 384-well microtiter plates using an Echo 550® acoustic liquid handler (Beckman Coulter™, Indianapolis, IN) and inoculated with bacteria to a final concentration of 10 6 CFU/mL, with a total assay volume of 25.25 µL. Plates were incubated with shaking for 24 hours at 37 ºC. Bacterial growth was measured by reading the OD 600 using an EnVision™ microplate reader (Revvity, Waltham, MA). The activity of the compounds was expressed as the percentage of bacterial growth inhibition, and it was calculated using the following normalization: Where, T 0 Sample is the absorbance of the strain growth in the presence of compound measured at time zero, T f Sample is the absorbance of the strain growth in the presence of compound measured at final time, T 0 Growth is the absorbance of the strain growth in the absence of compound measured at time zero, T f Growth is the absorbance of the strain growth in the absence of compound measured at final time, T 0 Blank is the absorbance of the broth medium (blank) measured at time zero, T f Blank : the absorbance of the broth medium (blank) measured at final time; T 0 is Time at 0 hour and T f is Time at 24 hours. Blank is composed of 25 µL of MHII and 0.25 µL of DMSO 20% Growth is composed of 25 µL of bacterial inoculum and 0.25 µL of DMSO 20%. Sample studied compound. The Genedata Screener software (Genedata, Inc., Basel, Switzerland) was used to process and analyse all the screening data. Their reproducibility and sensitivity were supported by the statistical values derived from all the experiments performed. Also, MIC 90% (Minimum Inhibitory Concentration required to inhibit 90% of the growth of a microorganism) value for every Dose Response Curve of reference antibiotic compounds (imipenem and colistin) was determined to assess consistent reproducible activity data within assay plates and between experiments. This software was used to calculate quality control parameters such as RZ’ factor (RZ’ factor ≥ 0.5), and signal/background ratio (S/B). The Z’factor predicts the robustness of an assay by considering the mean and standard deviation of both positive and negative controls 21 . The robust Z’ factor (RZ’ factor) is based on the Z’ factor, but standard deviations and means are replaced by the robust standard deviations and medians, respectively. in vitro susceptibility testing The minimum inhibitory concentration (MIC) of ENOblock and colistin were determined against all studied A. baumannii strains in two independent experiments using the broth microdilution method, following the standard guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 22 . A 5x10 5 CFU/mL inoculum of each strain was cultured in Luria-Bertani (LB) and cation-adjusted Mueller-Hinton broth, and then added to U-bottom microtiter plates (Deltalab, Spain) containing ENOblock or colistin. The plates were incubated for 18 hours at 37°C. Pseudomonas aeruginosa ATCC 27853 was used as the positive control strain. Time kill kinetic assays To determine the bactericidal activity, duplicate time-kill curves were performed for A. baumannii ATCC 179178 and Ab CR17 strains, as previously described 23 . An initial inoculum of 5x10 5 CFU/mL was added to LB in the presence of 1xMIC, 2xMIC and 4xMIC of ENOblock. A drug-free broth was evaluated in parallel as a control. Tubes of each condition were incubated at 37 ºC with shaking, and viable counts were determined by serial dilution at 0, 2, 4, 8, and 24 hours. Viable counts were determined by plating 100 µL of the control, test cultures, or the respective dilutions at the indicated times onto sheep blood agar plates (ThermoFisher, Spain). Plates were incubated for 24 hours at 37 ºC, and after colony counts, the log 10 of viable cells (CFU/mL) was determined. Bactericidal activity was defined as a reduction of ≥ 3 log 10 CFU/mL from the initial inoculum. Checkerboard assay The assay was performed on a 96-well plate in duplicate as previously described 23 . Colistin, imipenem, ceftazidime or tigecycline were two-fold serially diluted along the x-axis, whereas ENOblock was two-fold serially diluted along the y axis to create a matrix, where each well consists of a combination of both agents at different concentrations. Bacterial cultures grown overnight were then diluted in saline to 0.5 McFarland turbidity, followed by 1:50 further dilution LB and inoculation on each well to achieve a final concentration of approximately 5.5x10 5 CFU/mL. The 96-well plates were then incubated at 37°C for 18 hours and examined for visible turbidity. The fractional inhibitory concentration (FIC) of the colistin, imipenem, ceftazidime or tigecycline was calculated by dividing the MIC of colistin, imipenem, ceftazidime or tigecycline in the presence of ENOblock by the MIC of colistin, imipenem, ceftazidime or tigecycline alone. Similarly, the FIC of ENOblock was calculated by dividing the MIC of ENOblock in the presence of imipenem, ceftazidime or tigecycline alone. The FIC index was the summation of both FIC values. FIC index values of ≤ 0.5 and > 5 were interpreted as synergistic and non-synergistic, respectively. EIIP/AQVN filter Specific recognition and targeting between interacting biological molecules at distances > 5 Å were determined by the average quasi-valence number (AQVN) and the Electron-ion interaction potential (EIIP) derived from the general model pseudopotential 24 EIIP = 0.25 Z* sin(1.04 π Z*) (1) where Z* is the AQVN determined by: Z* = ∑m(ni Zi/N) (2) where Zi is the valence number of the ith atomic component, ni is the number of atoms of the ith component, m is the number of atomic components in the molecule, and N is the total number of atoms. EIIP values are computed using Equations (1) and (2) and are expressed in Rydberg units (Ry). AQVN and EIIP are unique physical properties that characterize long-range interactions between biological molecules among the 3300 molecular descriptors currently in use 25 . It has been shown that the EIIP and AQVN of organic molecules strongly correlate with their biological activity (mutagenicity, carcinogenicity, toxicity, antibiotic and cytostatic activity, etc.) 26 . Human cell culture HeLa cells were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), vancomycin (50 mg/L), gentamicin (20 mg/L), and amphotericin B (0.25 mg/L) (Invitrogen, Spain), and 1% HEPES in a humidified incubator with 5% CO 2 at 37°C. The HeLa cells were routinely passaged every 3 or 4 days. Immediately before infection, HeLa cells were washed three times with prewarmed PBS and further incubated in DMEM without FBS and antibiotics 27 . Adhesion and invasion assays HeLa cells were infected with A. baumannii ATCC 17978 and Ab CR17 strains at a concentration of 1×10 8 CFU/mL, in the absence and presence of 1xMIC of ENOblock at a multiplicity of infection (MOI) of 100. The infection was carried out for two hours with 5% CO 2 at 37°C in three independent experiments. After that, the infected HeLa cells were washed five times with pre-warmed PBS and lysed with 0.5% Triton X-100. Diluted lysates were plated onto LB agar and incubated at 37°C for 24 hours to enumerate the developed colonies and determine the number of bacteria that had attached to the HeLa cells 27 . In addition, to determine the number of colonies that entered inside the HeLa cells, the wells were washed with phosphate buffered saline and incubated for 30 minutes in the presence of DMEM plus gentamicin (256 µg/mL), in order to kill the bacteria present in the area. Then, the wells were washed with phosphate buffered saline to remove gentamicin. The number of colonies that entered inside HeLa cells was determined as described above 27 . Cellular toxicity of ENOblock HeLa cells and macrophages differentiated from THP-1 cells 28 were incubated with ENOblock at different concentration ranged from 0.5 to 256 mg/L for 24 hours with 5% CO 2 at 37°C. Prior the evaluation of the ENOblock cytotoxicity, HeLa and macrophage cells were washed three times with prewarmed PBS 1X. Subsequently, quantitative cytotoxicity was evaluated by measuring the mitochondrial reduction activity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously 29 . The percentage of cytotoxicity was calculated from the absorbance at 570 nm as follow: [(Absorbance 570 nm of treated cells/Absorbance 570 nm mean of untreated cells) × 100]. The cytotoxic concentration 50% (CC 50 ) value was determined using GraphPad Prism 9. Immunofluorescence Immunofluorescence assay was performed as described previously 30 . Briefly, the HeLa cells plated on coverslips were incubated with A. baumannii CR17 and E. coli MCR1 + strains for two hours, and later were incubated with ENOblock (0 and 1xMIC, 30 min) at 5% CO 2 and 37°C. Bacterial cells were removed and HeLa cells were washed five times with cold PBS. HeLa cells on the coverslips were fixed in methanol for 8 min at -20°C, permeabilized with 0.5% Triton X-100 and blocked with 20% pork serum in PBS. Primary antibodies: anti-OmpA of A. baumannii (ThermoFischer, Spain), mouse anti- E. coli (Abcam, Spain), and rabbit anti-human fibronectin (Merck, Spain) were used at dilution of 1:25, 1:25 and 1:50, respectively, in PBS containing 1% bovine serum albumin (BSA) for 2 hours. After washing with PBS, the coverslips were incubated with their respective secondary antibodies: Alexa488-conjugated goat anti-mouse IgG, and Alexa594-conjugated goat anti-rabbit IgG (Invitrogen, Spain) at dilution of 1:50, 1:50 and 1:100, respectively, in PBS containing 1% BSA for 1 hours. The fixed coverslips were incubated for 10 minutes at room temperature with DAPI (Applichem, Germany) (0.5 µg/mL), washed with PBS, mounted in fluorescence mounting medium “Prolong Diamond Antifade Mountant” (Invitrogen, Spain), and visualized using fluorescence microscopy Zeiss Axio Imager 2 (Zeiss, Germany). Bacterial cytological profiling Fluorescent microscopy Overnight cultures of A. baumannii were diluted 1:100 in LB broth and incubated on a roller at 30°C until the OD600 reached 0.2. ENOblock was added to the bacterial cultures at MIC levels prior to observation under a fluorescence microscope at various time points. After each timepoint, the cultures were stained with 2 µg/mL FM4-64, 2 µg/mL DAPI, and 0.5 µM SYTOX Green. The bacterial cells were then harvested by centrifugation at 6,000 x g for one minute and resuspended in 1/10 of the original volume. A small amount of the concentrated bacterial cultures was placed on an agarose pad (1.2% agarose in 10% LB broth) on concave glass slides for microscopy. Consistent experimental settings and imaging parameters were maintained throughout all experiments included in the statistical analysis of the antibiotic training sets. Image data analysis. The raw images from the fluorescent microscope were preprocessed using ImageJ software 31 , and cell features were extracted with CellProfiler 4.0 software 32 . After data extraction, the ENOblock-treated cell profiles were analyzed alongside antibiotic-treated cell profiles from previous studies using an analysis pipeline from previous research 33 . Briefly, the data was transformed using QuantileTransformer 34 , and outliers were removed using hierarchical density-based spatial clustering of applications with noise (HDBSCAN) 35 . This analysis utilized a morphological feature set from previous studies 33 . Finally, the dimension of the data set was reduced and visualized through data clustering using pairwise controlled manifold approximation (PaCMAP) 36 . Docking and molecular modelling The crystal structure of enolase C-terminal of A. baumannii was predicted by AlphaFold 37 . This target protein was modified using Autodock tools 1.5.6 software; including ligand and water removal, hydrogen addition, and incorporation of Kollman charges. The resultant files were saved in pdbqt format. The ligand "ENOblock" was downloaded from Pubchem ( https://pubchem.ncbi.nlm.nih.gov/ ) in SMILES format ( https://pubchem.ncbi.nlm.nih.gov/compound/24012277 ). Openbabel 3.1.1 software was used to create the 3D chemical structure, energy minimization, hydrogen atoms addition and establishment of a neutral pH. Resulting ligand was saved in MOL.2 format. Gasteiger charges computation for the ligand structure was performed using Autodock tools 1.5.6, with the output saved in pdbqt format. Autodock tools 1.5.6 was used for docking and subsequent analysis of the docking results. Generation of enolase knockout from A. baumannii ATCC 17978 To construct an enolase knockout from A. baumannii ATCC 17978, we followed the protocol described previously 29 , 38 . Briefly, an internal enolase 482-bp fragment obtained by PCR amplification with the primers enolase IntUp and enolase IntLw (Table S1) was cloned into pGEM-T (Promega, Spain) to give plasmid enolase-pGEM-T by using T4 DNA ligase (Promega, Spain). The resulting construct incorporated into E. coli DH5α was purified and electroporated into ATCC 17978 to knock out the enolase gene. Transformants were selected on LB agar plates containing 80 µg/mL ticarcillin. The enolase gene disruption within the resulting strain, designated Ab Δ eno , was confirmed by PCR using a combination of primers matching the upstream region of enolase gene and the pGEM-T Easy vector. Bacterial enolase activity assay Enolase activity was determined in three independent experiments following the instructions of Enolase Activity Assay kit (Merck, Spain). Briefly, a bacterial inoculum of 10 6 CFU/mL, after centrifugation, was incubated with a master mix composed by enolase substrate, peroxidase substrate, and the necessary converters for the colorimetric reaction at 25 ºC. Enolase enzymatic activity, reported in mU/mL, was measured at OD 570 nm every two-three minutes for one hour using a microtiter plate reader (Tecan Spark, Austria) and calculated using the specific formula: ΔA570 = (A570) final - (A570) initial , and the following equation: enolase Activity = (B × Sample Dilution Factor) / (Reaction Time × V) where: B = Amount (nmol) of H 2 O 2 generated between T initial and T final , Reaction Time = T fina l – T initial (minutes), and V = volume of sample (mL) added to the well Bacterial growth curves To determine the antibacterial of ENOblock against wild-type and enolase-defieicent A. baumannii , duplicate bacterial growth curves of the A. baumannii ATCC 17978 and its isogenic deficient in enolase (Ab Δ eno ) strain were performed in duplicate in 96-well plate (Deltlab, Spain). An initial inoculum of 5x10 5 CFU/mL was prepared in LB in the presence of 1x, 2x and 4x MIC of ENOblock. A drug-free broth was evaluated in parallel as a control. Plates were incubated at 37°C with shaking, and bacterial growth was monitored for 24 hours using a microtiter plate reader (Tecan Spark, Austria). Galleria mellonella infection model G. mellonella infection model with ATCC 17978 strain was established by haemocoel bacterial inoculation. Briefly, caterpillars obtained from Artroposfera (Toledo, Spain) were inoculated with 10 µL of the bacterial suspensions, which were incubated for 20–24 hours in LB at 37°C. The minimal bacterial lethal dose 100 (MLD100) and LD50 were determined by inoculating various groups of larvae (8 G. mellonella per group) with decreasing amounts of ATCC 17978 strains inocula from 10 6 to 10 2 CFU/mL, and monitoring the survival of the larvae for 7 days. Therapeutic efficacy of ENOblock in G. mellonella infection model The efficacy of the ENOblock treatment was tested in G. mellonella survival assay as previously described 39 . Caterpillars were injected by the 10 µL of suspension containing MLD of ATCC 17978. Treatment with 4xMIC of ENOblock was injected one hour post-infection. A group pf larvae injected with 10 µL of sterile PBS was included as control. After inoculation, the larvae were incubated at 37°C in the dark and death was assessed over 3 days. Statistical Analysis Group data are presented as means ± standard errors of the means (SEM). The student t -test was used to determine differences between means using the GraphPad Prism 9 (version 9.3.1; GraphPad Software, LLC.). For the G. mellonella survival model, a Kaplan-Meier test was performed to determine the difference between mortality rates. P < 0.05 was considered significant. RESULTS High-throughput screening for repurposing drugs as antimicrobial agents We developed and validated a high-throughput screen assay using the A. baumannii ATCC 17978 and E. coli ATCC 25922 strains, as well as their respective MDR strains (Fig. 1 A). In total, we screened 2,464 compounds from the EU-OPENSCREEN ECBL Pilot library. We identified 33 compounds (1.32% of the total compounds) with inhibitory activity of ≥ 70% against at least one or both MDR strains of A. baumannii and E. coli . Of these 33 compounds, 7 showed MICs of ≤ 100 µM against the reference and MDR strains of A. baumannii and E. coli (Fig. 1 A). Among these 7 compounds, ENOblock (Fig. 1 B) was chosen for further studies. This compound was active against the A. baumannii ATCC 17978 and E. coli ATCC 25922 strains, with AC 50 values of 23.57 µM and 46.86 µM, respectively (Fig. 1 C). ENOblock is active against A. baumannii To confirm the activity of ENOblock against clinical isolates of A. baumannii , ENOblock was tested against 14 and 18 colistin-resistant and carbapenem-resistant A. baumannii isolates. The results of the MICs tests are displayed in Table 1 and Table 2 . The MICs ranged from 8 to 32 mg/L and 16 to 32 mg/L for ENOblock against colistin and carbapenem-resistant A. baumannii , respectively. The reference strain ATCC 17978 present an ENOblock MIC of 8 mg/L. The MIC 50 and MIC 90 concentrations, which represent the concentration effective for 50 and 90% of the isolates tested, respectively, for ENOblock against colistin and carbapenems-resistant isolates were 16 and 32 mg/L, respectively. However, the MIC 50 and MIC 90 for colistin were 256 and > 256 mg/L, and for carbapenems were 16 and 64 mg/l (Table 1 ). Of note, the ENOblock MIC 90 is three times below the CC 50 of ENOblock in HeLa and macrophage cells (data not shown). Table 1 Antibacterial activity of ENOblock in colistin-resistant A. baumannii strains. Strains Colistin MIC (mg/L) ENOblock MIC (mg/L) #1 256 16 #10 > 256 16 #11 256 16 #14 256 16 #16 > 256 16 #17 64 16 #19 > 256 32 #20 > 256 8 #21 256 8 #22 > 256 16 #24 64 16 #99 > 256 16 #113 > 256 16 Ab CR17 64 32 MIC 50 256 16 MIC 90 > 256 32 Table 2 Antibacterial activity of ENOblock in carbapenem-resistant A. baumannii strains. Strains Imipenem/meropenem MIC (mg/L) ENOblock MIC (mg/L) #17 8 16 #37 16 16 #40 > 64 16 #53 64 16 #286 16 16 #288 32 16 #289 8 16 #295 8 16 #298 16 32 #299 16 16 #405 32 32 #410 32 32 #414 32 32 #416 32 32 #417 16 16 #440 8 16 #441 8 16 #448 8 32 MIC 50 16 16 MIC 90 64 32 Using time-course assays, we evaluated the bactericidal activity of ENOblock against ATCC 17978 and Ab CR17 strains. Figure 2 A illustrates that ENOblock (2x and 4xMIC for ATCC 17978 strain) exhibited bactericidal effect after 2, 4 and 8 hours, reducing the bacterial count by over 3 log 10 CFU/mL compared to 0 hours. For the Ab CR17 strain, ENOblock (1x, 2x and 4xMIC for Ab CR17 strain) demonstrated a bactericidal effect after 2, 4 and 8 hours by reducing the bacterial count by over 3 log 10 CFU/mL, compared to 0 hours. It is well known that the development of new repurposed drugs includes the assessment of the presence of synergy with clinically used antibiotics. To this end, we conducted a virtual screening of ENOblock in combination with different antibiotics (colistin, imipenem, ceftazidime and tigecycline) using the EIIP/AQVN criterion to overcome bacterial resistance (Table S2). Figure 2 B suggests that colistin and ENOblock with similar electronic properties, as indicated by their EIIP and AQVN values, tend to exhibit synergistic effects. Conversely, no synergistic activity was observed between the rest of antibiotics and ENOblock with different electronic properties. To confirm experimentally the in silico synergistic effect of ENOblock with colistin against A. baumannii , checkerboard assay was performed. This assay indicated that ENOblock had a synergistic effect with colistin by enhancing the activity of colistin against ATCC 17978 and Ab CR17, resulting in an FIC index (FICI) of ≤ 0.5. In contrast, the combination of ENOblock with other antibiotics such as imipenem, ceftazidime and tigecycline did not increase their activities, yielding a FICI > 0.5 (Fig. 2 C). ENOblock affects the A. baumannii -host interaction To evaluate the effect of ENOblock on the interaction between A. baumannii and host cells, we studied the adherence and invasion of the ATCC 17978 and Ab CR17 strains on HeLa cells for two hours in the presence of ENOblock. We found that treatment with ENOblock at 1xMIC reduced the counts of adherent ATCC 17978 and Ab CR17 strains on HeLa cells by 47% ( P < 0.05) and 31% ( P < 0.05), respectively. Notably, a more significant reduction was observed in the invasion of both strains, with ENOblock treatment reducing invasive counts by 76% ( P < 0.01) and 46% ( P < 0.05), respectively (Fig. 3 A). Furthermore, immunostaining of infected HeLa cells with the ATCC 17978 and Ab CR17 strains, pretreated with ENOblock, showed a significant reduction in A. baumannii attachment to HeLa cells (Fig. 3 B). ENOblock inhibits the growth of A. baumannii via distinct mechanism of action We employed the fluorescence microscopy-based BCP technique, as previously applied to A. baumannii 33 , 40 , 41 , to investigate the mechanism of action of ENOblock against ATCC 17978 strain. The morphological changes induced by ENOblock were compared with those of antibiotics targeting major cellular pathways, including ciprofloxacin CIP (DNA replication), rifampicin RIF (RNA transcription), minocycline MIN (protein translation), piperacillin PIP and meropenem MER (cell wall synthesis), and colistin CST (membrane integrity). BCP results showed that ENOblock-treated cells exhibit unique morphological changes compared to those of antibiotic controls. In particular, membrane blebs were observed, as was the high SYTOX green signal of ENOblock-treated cells (Fig. 3 C), indicating the loss of membrane integrity. However, the ENOblock-treated cells showed different morphological changes from CST-treated cells, a membrane integrity control, indicating that ENOblock interfere with bacterial membrane integrity but possibly in a manner different from CST. Consistent with these differences, the image analysis profiles of ENOblock-treated cells clustered separately from those of untreated, other control antibiotics (Fig. 3 D). It is conceivable that ENOblock inhibits pathways that are distinct from those targeted by the comparator antibiotics, which collectively represent the most common modes of antibacterial action, in a manner similar to previous studies that have observed distinct profiles 29 , 42 – 44 . Altogether, the results showed that the ENOblock profile is distinct from the six antibiotic profiles. ENOblock exhibits rapid permeabilization activity against A. baumannii The observation of a higher proportion of ENOblock-treated cells displaying high SYTOX Green intensity prompts us to consider the possibility of observing this impact at earlier time intervals, as membrane permeabilization often happens swiftly within minutes. Prior studies utilizing BCP demonstrated that a compound with the ability to disrupt membranes could impact the bacterial membrane in a mere 10 minutes timeframe 39 , 41 . Consequently, we conducted a temporal examination of SYTOX green staining on cells treated with ENOblock for 10, 30, and 60 minutes. The findings indicated that cells treated with ENOblock exhibited a notable rise in SYTOX intensity just 10 minutes after treatment (Fig. 3 E, F), in comparison to the control group that did not receive treatment ( P < 0.01). Although the fluorescence levels continued to increase after 30 minutes, this rise was not statistically significant (Fig. 3 F), indicating that the cells reached a saturation point with SYTOX Green after 10 minutes. Hence, the results indicates that ENOblock rapidly compromises the rigidity of the bacterial cell envelope, potentially resulting in cell death. ENOblock acts on A. baumannii through the inhibition of enolase In order to shed light on the ENOblock mechanism of action, we docked ENOblock in the C-terminal domain of A. baumannii enolase. ENOblock exhibited a high docking score. The most stable pose shows that ENOblock binds to Ser371 and Asp207 amine acids through 3 hydrogen bonds (Fig. 4 A). To confirm that enolase is the potential target of ENOblock, we generated an enolase-deficient mutant. Deletion of the enolase gene in the ATCC17978 strain (Δ eno ) first abolished completely the enolase activity (Fig. 4 B) and subsequently increased the ENOblock MIC from 8 to 32 mg/L (Fig. 4 C). Furthermore, we examined the antibacterial activity of ENOblock against the ATCC 17978 and Δ eno strains. Figure 4 D reveals that the ATCC 17978 strain exhibits rapid growth, reaching an OD of 1 within the first 4 hours. However, a significant disparity in growth is observed between the untreated cells and the cells treated with ENOblock, particularly at higher compound concentrations (16 and 32 mg/L). A different trend of growth inhibition is observed in the Δ eno strain, which shows a higher OD value compared with A. baumannii ATCC 17978 strain in the presence of ENOblock treatment (Fig. 4 E). This difference in growth can be attributed to the resistance of the mutant strain to the ENOblock, as the absence of enolase may hinder the compound’s ability to exert its effect, as indicated by the findings of the molecular docking study. ENOblock presents therapeutic efficacy in vivo To confirm the in vitro effect of ENOblock in monotherapy and in combination with colistin against A. baumannii , and to study this efficacy in a complete organism, we moved to an invertebrate model of infection by A. baumannii . First, we determined the virulence of the ATCC 17078 strain after haemocoel administration in G. mellonella . The mortality rates of animals were inoculum concentration dependent. LD 50 and MLD 100 for the ATCC 17978 strain were 10 2 and 1x10 5 CFU/mL, respectively. Subsequently, in a G. mellonella model of infection, we administered ENOblock (32 mg/L) to animals after haemoceol administration of an MLD 100 of the ATCC 17978 strain (Fig. 5 C). Animals receiving treatment with ENOblock showed significantly a greater increase in survival compared with untreated animals ( P < 0.01) (Fig. 5 C). DISCUSSION The emergence of MDR A. baumannii has led to the use of colistin as a last resort for treating severe infections caused by this pathogen. Although colistin resistance is still uncommon, its emergence and spread are regarded as concerns to world health 45 . Due to the antibacterial effects of various anticancer drugs on A. baumannii 15 – 17 , 46 , we hypothesized that ENOblock, identified in this study after a HTS of the EU-OPENSCREEN library, might exhibit strong antibacterial activity against colistin- or carbapenem-resistant A. baumannii . After initial antimicrobial confirmation of ENOblock, we additionally tested its susceptibility against 32 clinical isolates of A. baumannii resistant to colistin or carbapenems (Tables 1 and 2 ). The ENOblock MIC 90 is 32 mg/L, which is two to more than six times lower than the MIC 90 of carbapenem and colistin, respectively. This MIC 90 value falls within the range of other known antibiotics such as amikacin, amoxicillin-clavulanic acid, ceftazidime-avibactam, and fosfomycin, among others 47 . It is noteworthy that ENOblock demonstrated similar activity against MDR E. coli (data not shown), consistent with previously published data on the antibacterial activity of the anticancer drug family tamoxifen and its metabolites used in earlier studies 16 , 17 , 46 . Specific AQVN/EIIP domains, combined with structural properties, can serve as effective filters for the virtual screening of molecular libraries to identify new drug candidates, including new antibiotics. Using molecular descriptors, the EIIP and AQVN we have proposed suitable antibiotics for treating MDR bacterial infections 48 . In this study, we analyzed the electronic properties of ENOblock and antibiotics to which A. baumannii ATCC 17978 is sensitive. Our analysis suggests that antimicrobials with similar electronic properties tend to act synergistically. However, the limited number of molecules analyzed restricts our ability to establish a criterion for predicting synergy in A. baumannii . Moreover, studies show that the molecular mechanisms underlying such synergistic effects remain not fully understood 49 . Nonetheless, the presented results with the observed tendency for ENOblock and colistin, to exhibit synergy suggest that similar electronic properties may contribute to effective antibacterial combinations. Small molecules with similar AQVN and EIIP values have previously been shown to interact with the common therapeutic target 50 , 51 . The antibacterial activity of ENOblock at 1x, 2x, and 4xMIC against the colistin-resistant strain (Ab CR17) is higher than against the colistin-susceptible reference strain (ATCC 17978). This result may be related to differences in the cell wall structure of the two strains, where colistin-resistant strains of A. baumannii are more permeable than colistin-susceptible strains 23 , 52 , 53 . The antimicrobial activity of ENOblock identified in this study suggests promising potential that warrants further exploration in vivo after determining its pharmacokinetic parameters. However, in vitro bacterial growth showed a progressive regrowth of the ATCC 17978 strain after treatment with ENOblock at 1xMIC, suggesting that this strain may have acquired resistance to this compound. It is worth noting that the MIC of ENOblock against the ATCC 17978 strain in these time-kill assay conditions is 8 mg/L, which is below the 2x and 4xMIC of ENOblock. Further investigations, including the determination of its concentration during the time-kill assay, are necessary to better understand the regrowth of this strain in the presence of ENOblock. Additionally, BCP analysis showed that ENOblock-treated A. baumannii exhibits distinct morphological changes compared to comparator antibiotics (Fig. 3 C, D), suggesting that ENOblock inhibits pathways different from those targeted by the comparator antibiotics. Membrane-disruptive agents can be classified into various subcategories, each exhibiting a unique structure on BCP. Nevertheless, the BCP profile of ENOblock-treated cells observed in this study did not precisely correspond to any previously described profiles 29 , 40 – 43 . Consequently, the specific target on the membrane for ENOblock could not be determined, but the presence of SYTOX Green indicates that ENOblock can permeabilize membranes. Therefore, future studies should investigate the precise locations on the bacterial membrane where ENOblock is active. It is widely known that ENOblock inhibits enolase activity in eukaryotic cells 54 , and enolase, a cytoplasmic glycolytic enzyme, is a key component of the RNA degradosome in Gram-negative bacteria such as E. coli and Pseudomonas aeruginosa 55 . For this reason, we decided to conduct a preliminary computational study to determine if ENOblock could potentially bind to enolase in A. baumannii . As a result, ENOblock exhibited a better docking score. Deletion of enolase gene in A. baumannii increased the ENOblock MIC four-fold and increased bacterial growth in the presence of ENOblock, suggesting that enolase could be a potential target of ENOblock in A. baumannii . Importantly, two studies have reported that enolase also resides on the cell wall outer membrane of E. coli and P. aeruginosa 56 , 57 and mediates the binding of P. aeruginosa to plasminogen on host cells 57 . In this study, we showed that ENOblock reduces the interaction of A. baumannii with host cells, possibly by affecting the plasminogen-binding activity of A. baumannii outer membrane enolase 58 . Consequently, the possibility of multi-targeting, with ENOblock binding to both cytosolic and membranal enolase, might contribute to its overall activity, which is an attractive aspect of ENOblock's antibacterial properties. We have demonstrated through various assays that ENOblock can be repurposed as an antibacterial agent. However, the ultimate goal of this compound is to achieve good therapeutic efficacy in animal models of infection. So far, ENOblock at 32 mg/L has been able to increase animal survival in presence of A. baumannii . This result is consistent with previous observations that deletion of enolase gene abolishes the virulence of P. aeruginosa in a murine acute pneumonia model 59 . In summary, this drug discovery approach ought to be viewed as the first step in creating a brand-new class of antimicrobial drugs. The efficacy of ENOblock can be investigated further by combining this newly repurposed compound with clinically used antibiotics (like colistin) in in vivo experiments, with the aim of reducing mortality and improving therapeutic efficacy in cases of severe infections, even with the current antimicrobial therapies. Declarations Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author contributions I.M.P., A.M.R., M.C., P.S., T.S., V.V. & A.H. methodology, investigation, formal analysis. A.P.P, S.G., O.G. A.H. & P.N. writing-review and editing, methodology, investigation. Y.S. writing-review and editing, supervision, funding acquisition, conceptualization. Acknowledgments This research was funded by the Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación, Fondo Europeo de Desarrollo Regional, MCIN/AEI/ 10.13039/501100011033/FEDER , UE (Grant PID2022-136357OBI00), and (Grant CEX2020-001088-M-20-5), by the Consejería de Universidad, Investigación e Innovación de la Junta de Andalucía (Grant ProyExcel_00116), by the National Research Council of Thailand (NRCT) and Mahidol University: N42A650368, and by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Grant number 451-03-66/2024-03/200017). We acknowledge EU-OPENSCREEN ERIC for providing its compound collection and Fundación MEDINA HTS antimicrobial screening platform to support the discovery of the antibacterial activity of the compound described in the presented work. This article is based upon work from COST Action EURESTOP, CA21145, supported by COST (European Cooperation in Science and Technology). A.M.R is supported by a doctoral fellowship PRE2022-104318, from the Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación. Data availability statement The data supporting the findings of this study are available from the corresponding author upon reasonable request. References Morris S, Cerceo E (2020) Trends, epidemiology, and management of multi-drug resistant gram-negative bacterial infections in the hospitalized setting. Antibiotics 9:196 Balasubramanian R et al (2023) Global incidence in hospital-associated infections resistant to antibiotics: An analysis of point prevalence surveys from 99 countries. PLoS Med 20:e1004178 Yelin I, Kishony R, SnapShot (2018) Antibiotic resistance. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5059044","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":353568195,"identity":"308ba246-a256-494e-963d-9fd9c2eadad3","order_by":0,"name":"Younes Smani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAkklEQVRIiWNgGAWjYLCCDyTrYJxBshZmHpKUG5w/nfjZtu2OHAN7+wMitdzI3Syd2/bMmIHnjAGxWng3ALUcTmyQyCHaYWc3/7YEaZF/TqzDDuRuk2YE28JApMMkb+Rus+w598yYjSeHSC18QIfd+FF2R46f/TiRDlM4AKYOMLARpx4I5BugWkbBKBgFo2AU4AQAvJcuZR7Bwh4AAAAASUVORK5CYII=","orcid":"","institution":"Andalusian Center of Developmental Biology, CSIC, University of Pablo de Olavide - Seville (Spain)","correspondingAuthor":true,"prefix":"","firstName":"Younes","middleName":"","lastName":"Smani","suffix":""},{"id":353568196,"identity":"deab345c-c1bb-47dd-87b8-84c4194a1167","order_by":1,"name":"Irene Molina Panadero","email":"","orcid":"","institution":"Centro Andaluz de Biología del Desarrollo","correspondingAuthor":false,"prefix":"","firstName":"Irene","middleName":"Molina","lastName":"Panadero","suffix":""},{"id":353568197,"identity":"b015c7bf-d7ec-4775-86f5-c799a3e58bca","order_by":2,"name":"Antonio Moreno Rodríguez","email":"","orcid":"","institution":"Andalusian Center of Developmental Biology, CSIC, University of Pablo de Olavide - Seville (Spain)","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"Moreno","lastName":"Rodríguez","suffix":""},{"id":353568198,"identity":"9ae4e581-d82c-446e-862f-a267e3c6d4b1","order_by":3,"name":"Mercedes de la Cruz","email":"","orcid":"","institution":"Fundación MEDINA, Parque Tecnológico Ciencias de la Salud, Granada, España","correspondingAuthor":false,"prefix":"","firstName":"Mercedes","middleName":"de la","lastName":"Cruz","suffix":""},{"id":353568199,"identity":"ea7c54f4-bddb-47bc-8377-ec058bffb37f","order_by":4,"name":"Pilar Sánchez","email":"","orcid":"","institution":"Fundación MEDINA, Parque Tecnológico Ciencias de la Salud, Granada, España","correspondingAuthor":false,"prefix":"","firstName":"Pilar","middleName":"","lastName":"Sánchez","suffix":""},{"id":353568200,"identity":"408b0962-7b52-41d2-9848-30b980d75aec","order_by":5,"name":"Thanadon Samernate","email":"","orcid":"","institution":"Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhon Pathom , Thailand.","correspondingAuthor":false,"prefix":"","firstName":"Thanadon","middleName":"","lastName":"Samernate","suffix":""},{"id":353568201,"identity":"ba217268-d6f8-4ad7-9927-e6a2910e034e","order_by":6,"name":"Antonio Pérez-Pulido","email":"","orcid":"https://orcid.org/0000-0003-3343-2822","institution":"Andalusian Centre for Developmental Biology (CABD, UPO-CSIC-JA). 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(A) Hit identification workflow using the EU-OPENSCREEN library. *Screening of compounds with ≥ 70% activity against 1 or 2 MDR pathogens. **Screening of compounds with MIC≤100 μM w/o previous antibacterial activity. (B) Chemical structure of ENOblock. (C) Confirmation of ENOblock activity with the primary screening sample in dose-response mode against A. baumannii ATCC 17978 and E. coli ATCC 25922 strains. AC50: Concentration at which 50% of maximum activity is observed.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5059044/v1/a7bdb75beac6f7167161d00a.png"},{"id":73049815,"identity":"36c5464a-6077-4c35-bcb1-521da2522e20","added_by":"auto","created_at":"2025-01-06 09:23:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":101666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eENOblock is active against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. baumannii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein monotherapy and in combination with colistin.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Time-kill curves of \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 and Ab CR17 strains in the presence of 1x and 1x, 2x and 4xMIC ENOblock\u003cstrong\u003e \u003c/strong\u003efor 24 hours. (\u003cstrong\u003eB\u003c/strong\u003e) Schematic presentation of the EIIP/AQVN criterion for the selection of ENOblock and colistin combination (green-penicillins, blue-carbapenems, red-quinolones). (\u003cstrong\u003eC\u003c/strong\u003e) Representative heat plots of microdilution checkerboard assay for the combination of ENOblock and colistin against ATCC 17978 and Ab CR17 strains. AQVN: Average quasi-valence number, EIIP: Electron-ion interaction potential.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5059044/v1/fe5822145290312d1ac29f39.png"},{"id":73049819,"identity":"63508aab-178f-4086-ae16-f1274f466d13","added_by":"auto","created_at":"2025-01-06 09:23:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":299644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eENOblock exhibit antibacterial activity against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. baumannii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e via distinct mechanism of action and displays rapid permeabilization activity\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Analysis of \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 and Ab CR17 strains adhesion/invasion into HeLa cells with (1xMIC) and without ENOblock treatment. The data are presented as means ± SEM, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05: treatment vs no treatment; two-tailed Student’s t-test. (\u003cstrong\u003eB\u003c/strong\u003e) Immunostaining of fibronectin of HeLa cells (magenta) and ATCC 17978 and Ab CR17 strains (green) pretreated with ENOblock (0x and 1xMIC), after bacterial adherence for two hours, was performed by specific primary antibodies against both strains and their respective secondary antibodies. Blue staining shows the location of HeLa cell nuclei. (\u003cstrong\u003eC\u003c/strong\u003e) Representative images and (\u003cstrong\u003eD\u003c/strong\u003e) a PaCMAP plot of \u003cem\u003eA. baumannii\u003c/em\u003e cells treated with 1x MIC of ENOblock and antibiotic controls. In all images, cell membranes are stained with FM4-64 (red), DAPI (blue), and SYTOX green (green). Scale bar represents 1 μM\u003cem\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003c/em\u003e(\u003cstrong\u003eE\u003c/strong\u003e)\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eRepresentative images of\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eA. baumannii\u003c/em\u003e cells treated with ENOblock for 10, 30 and 60 minutes, and then stained with FM4-64 (red) and SYTOX Green (green). Scale bar represents 1 μm. (\u003cstrong\u003eF\u003c/strong\u003e) A graph showing differences in SYTOX Green intensities between untreated and ENOblock-treated cells at different timepoints. Values represent mean intensities ± SEM of individual nucleoids from single cells, per condition. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; two-tailed Student’s t-test, n.s.; not statistically significant.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5059044/v1/4f8e38249f2588306f5c497f.png"},{"id":73049818,"identity":"81faa934-1b95-47f9-a84c-5b30de951327","added_by":"auto","created_at":"2025-01-06 09:23:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":66316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eENOblock acts on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. baumannii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e through the inhibition of enolase. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Structural models generated by docking of ENOblock into the C-domain of \u003cem\u003eA. baumannii\u003c/em\u003e enolase. ENOblock is displayed as sticks. ΔG: Glide score. (\u003cstrong\u003eB\u003c/strong\u003e) Enolase activity in determined in \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 and Δ\u003cem\u003eeno \u003c/em\u003estrains. The data are presented as means ± SEM, and student t-test was used for statistical analysis. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ATCC 17978 vs Δ\u003cem\u003eeno\u003c/em\u003e; two-tailed Student’s t-test. (\u003cstrong\u003eC\u003c/strong\u003e) MIC of ENOblock against \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 vs Δ\u003cem\u003eeno\u003c/em\u003e strains. (\u003cstrong\u003eD\u003c/strong\u003eand \u003cstrong\u003eE\u003c/strong\u003e) Bacterial growth curve plots of \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 vs Δ\u003cem\u003eeno\u003c/em\u003e strains in the absence and presence of ENOblock treatment at different concentrations.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5059044/v1/41d4cffe3e3f1ca227d0bddb.png"},{"id":73049829,"identity":"0d045d19-89cd-4edd-8f6e-af8b8439bcbb","added_by":"auto","created_at":"2025-01-06 09:23:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":38908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eENOblock displays efficacy in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eG. mellonella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e model of infection\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Experimental design for \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 strain lethal doses determination. (\u003cstrong\u003eB\u003c/strong\u003e) Seven days of mortality monitoring in \u003cem\u003eG. mellonella\u003c/em\u003e administered with different inoculum of \u003cem\u003eA. baumannii \u003c/em\u003eATCC 17978 strain (n = 8 per group). (\u003cstrong\u003eC\u003c/strong\u003e) Experimental design for \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 strain infection and treatment. (\u003cstrong\u003eD\u003c/strong\u003e) Three days of mortality monitoring in \u003cem\u003eG. mellonella\u003c/em\u003e administered with MDL\u003csub\u003e100\u003c/sub\u003e (10\u003csup\u003e5\u003c/sup\u003e CFU/mL) \u003cem\u003eA. baumannii \u003c/em\u003eATCC 17978 and treated or not with ENOblock (32 mg/L; n = 8 per group). \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, treatment vs no treatment; Kaplan-Meier test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5059044/v1/7d991dffa2f8a526e7670aea.png"},{"id":73053237,"identity":"60daa5f8-9dc3-4cc8-80d6-e65047de0005","added_by":"auto","created_at":"2025-01-06 09:47:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1477935,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5059044/v1/55ea5ce2-4a37-43fc-9209-6f4454fdd702.pdf"},{"id":73049814,"identity":"1bbe0b6e-87b2-4699-b709-524b5db61ed6","added_by":"auto","created_at":"2025-01-06 09:23:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15748,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1andS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5059044/v1/7052bbdd896d45f7cb91ac03.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A novel antibiotic class targeting the enolase of Acinetobacter baumannii","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eGram-negative bacteria (GNB) are a significant concern in healthcare settings due to their ability to cause a wide range of infections such as pneumonia, bloodstream infections, wound or surgical site infections, and meningitis\u003csup\u003e \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e \u003c/sup\u003e, that are often difficult to treat. GNB infections, especially hospital acquired ones, pose a significant burden on healthcare systems worldwide, with reported costs of \u003cspan\u003e$\u003c/span\u003e136\u0026nbsp;million per year\u003csup\u003e \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e \u003c/sup\u003e, requiring ongoing efforts in surveillance, infection prevention and control, antibiotic stewardship, and research into new treatment options to mitigate their impact.\u003c/p\u003e \u003cp\u003eOne of the main difficulties in tackling GNB is their high efficiency in acquiring antimicrobial resistance (AMR) encoded by genomic, transcriptomic and proteomic changes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Compounding the problem of AMR with reported high number of deaths associated with bacterial AMR and/or attributable to bacterial AMR is the immediate threat of a reduction in the discovery and development of new antibiotics. The World Health Organization (WHO)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and other European institutions have recently underscored the dangers posed by these infections\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Consequently, a perfect storm is converging regarding these infections: increasing antimicrobial resistance with a decreased new drug development\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This context is likely the best example of the purported \u0026ldquo;Post-Antibiotic Era\u0026rdquo;, with relevance even in non-specialized media. It is clear that new policies and actions are necessary to avoid the forecasts for 2050 that attribute ten million deaths worldwide to antimicrobial resistance\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Especially for pathogens like \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e that is reported with continuous increase in AMR and are accounted for most reported associated diseases and deaths\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eApproaches using computational methods and high-throughput screening (HTS) have recently been developed for antibiotic discovery\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. For example, screening small-molecule libraries has revealed new antimicrobial agents that belong to existing or new antibiotic classes\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Recently, HTS studies have been developed to discover repurposed drugs for antimicrobial treatments\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Previously, we identified anticancer drugs such as Selective Estrogen Receptor Modulators - specifically tamoxifen and its metabolites - as new agents for the treatment of \u003cem\u003eA. baumannii\u003c/em\u003e\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this work, we report the identification of ENOblock, an anticancer drug, as a novel antibiotic class. We computationally and experimentally validated that ENOblock synergizes with the last resort antibiotic, the colistin. Additionally, we identified enolase as the potential bacterial target for ENOblock. The \u003cem\u003ein silico\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e antibacterial activity of ENOblock translated into potent \u003cem\u003ein vivo\u003c/em\u003e efficacy in animal models of infection. Collectively, these preclinical data could support the selection of ENOblock as a promising candidate for antimicrobial development with the potential to address the urgent threat of infections caused by \u003cem\u003eA. baumannii\u003c/em\u003e.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Strains\u003c/h2\u003e \u003cp\u003eA total of 32 clinical strains of \u003cem\u003eA. baumannii\u003c/em\u003e colistin-resistant (n\u0026thinsp;=\u0026thinsp;14)\u003csup\u003e18,19\u003c/sup\u003e or carbapenem-intermediate/resistant (n\u0026thinsp;=\u0026thinsp;18) were collected from the \"II Spanish Study of \u003cem\u003eA. baumannii\u003c/em\u003e GEIH-REIPI 2000\u0026ndash;2010\" multicenter study (GenBank Bioproject PRJNA422585) and the reference strain ATCC 17978 were used in this study.\u003c/p\u003e \u003cp\u003eHTS was performed with two reference strains of \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC 17978 and ATCC 25922, respectively) and four well-characterized, clonally unrelated clinical strains: \u003cem\u003eA. baumannii\u003c/em\u003e Ab9 (ST672), colistin- and tigecycline-susceptible; MDR \u003cem\u003eA. baumannii\u003c/em\u003e Ab186 (ST208), colistin-susceptible, tigecycline-resistant; \u003cem\u003eE. coli\u003c/em\u003e C1-7-LE (ST8671), colistin- and tigecycline-susceptible; and MDR \u003cem\u003eE. coli\u003c/em\u003e MCR1\u003csup\u003e+\u003c/sup\u003e (ST6108), colistin-resistant, tigecycline-susceptible\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEU-OPENSCREEN library and HTS validation\u003c/h3\u003e\n\u003cp\u003eEU-OPENSCREEN provided a subset of 2,464 bioactive compounds from the ECBL pilot library (EU-OPENSCREEN: European Chemical Biology Library - Pilot Library) as 10 mM stock solutions in 100% DMSO. The library was screened in duplicate at a final concentration of 100 \u0026micro;M per well (1% DMSO). The antibacterial single-concentration HTS and dose-response susceptibility assays were conducted in 384-well plates, with bacterial cell density measured by optical density at 600 nm (OD600).\u003c/p\u003e \u003cp\u003eA starting inoculum of 10\u003csup\u003e6\u003c/sup\u003e colony-forming units (CFU)/mL was used, with an incubation time of 24 hours for the \u003cem\u003eA. baumannii\u003c/em\u003e (ATCC 17978, Ab9, and Ab186) strains and \u003cem\u003eE. coli\u003c/em\u003e (ATCC 25922, C17LE, and MCR1\u003csup\u003e+\u003c/sup\u003e) strains. Imipenem and colistin were used as internal controls for \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e strains, respectively.\u003c/p\u003e \u003cp\u003eCompounds were distributed in duplicate on separate 384-well microtiter plates using an Echo 550\u0026reg; acoustic liquid handler (Beckman Coulter\u0026trade;, Indianapolis, IN) and inoculated with bacteria to a final concentration of 10\u003csup\u003e6\u003c/sup\u003e CFU/mL, with a total assay volume of 25.25 \u0026micro;L. Plates were incubated with shaking for 24 hours at 37 \u0026ordm;C. Bacterial growth was measured by reading the OD\u003csub\u003e600\u003c/sub\u003e using an EnVision\u0026trade; microplate reader (Revvity, Waltham, MA). The activity of the compounds was expressed as the percentage of bacterial growth inhibition, and it was calculated using the following normalization:\u003c/p\u003e \u003cp\u003e\u003cimg 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\" width=\"496\" height=\"94\"\u003e\u003c/p\u003e\u003cp\u003eWhere, \u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003e0 Sample\u003c/b\u003e\u003c/sub\u003e is the absorbance of the strain growth in the presence of compound measured at time zero, \u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003ef Sample\u003c/b\u003e\u003c/sub\u003e is the absorbance of the strain growth in the presence of compound measured at final time,\u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003e0 Growth\u003c/b\u003e\u003c/sub\u003e is the absorbance of the strain growth in the absence of compound measured at time zero, \u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003ef Growth\u003c/b\u003e\u003c/sub\u003e is the absorbance of the strain growth in the absence of compound measured at final time, \u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003e0 Blank\u003c/b\u003e\u003c/sub\u003e is the absorbance of the broth medium (blank) measured at time zero, \u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003ef Blank\u003c/b\u003e\u003c/sub\u003e : the absorbance of the broth medium (blank) measured at final time; \u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003e0\u003c/b\u003e\u003c/sub\u003e is Time at 0 hour and \u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003ef\u003c/b\u003e\u003c/sub\u003e is Time at 24 hours.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBlank\u003c/strong\u003e \u003cp\u003eis composed of 25 \u0026micro;L of MHII and 0.25 \u0026micro;L of DMSO 20%\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGrowth\u003c/strong\u003e \u003cp\u003eis composed of 25 \u0026micro;L of bacterial inoculum and 0.25 \u0026micro;L of DMSO 20%.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSample\u003c/strong\u003e \u003cp\u003estudied compound.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe Genedata Screener software (Genedata, Inc., Basel, Switzerland) was used to process and analyse all the screening data. Their reproducibility and sensitivity were supported by the statistical values derived from all the experiments performed. Also, MIC 90% (Minimum Inhibitory Concentration required to inhibit 90% of the growth of a microorganism) value for every Dose Response Curve of reference antibiotic compounds (imipenem and colistin) was determined to assess consistent reproducible activity data within assay plates and between experiments. This software was used to calculate quality control parameters such as RZ\u0026rsquo; factor (RZ\u0026rsquo; factor\u0026thinsp;\u0026ge;\u0026thinsp;0.5), and signal/background ratio (S/B). The Z\u0026rsquo;factor predicts the robustness of an assay by considering the mean and standard deviation of both positive and negative controls\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The robust Z\u0026rsquo; factor (RZ\u0026rsquo; factor) is based on the Z\u0026rsquo; factor, but standard deviations and means are replaced by the robust standard deviations and medians, respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003esusceptibility testing\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe minimum inhibitory concentration (MIC) of ENOblock and colistin were determined against all studied \u003cem\u003eA. baumannii\u003c/em\u003e strains in two independent experiments using the broth microdilution method, following the standard guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. A 5x10\u003csup\u003e5\u003c/sup\u003e CFU/mL inoculum of each strain was cultured in Luria-Bertani (LB) and cation-adjusted Mueller-Hinton broth, and then added to U-bottom microtiter plates (Deltalab, Spain) containing ENOblock or colistin. The plates were incubated for 18 hours at 37\u0026deg;C. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853 was used as the positive control strain.\u003c/p\u003e\n\u003ch3\u003eTime kill kinetic assays\u003c/h3\u003e\n\u003cp\u003eTo determine the bactericidal activity, duplicate time-kill curves were performed for \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 179178 and Ab CR17 strains, as previously described\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. An initial inoculum of 5x10\u003csup\u003e5\u003c/sup\u003e CFU/mL was added to LB in the presence of 1xMIC, 2xMIC and 4xMIC of ENOblock. A drug-free broth was evaluated in parallel as a control. Tubes of each condition were incubated at 37 \u0026ordm;C with shaking, and viable counts were determined by serial dilution at 0, 2, 4, 8, and 24 hours. Viable counts were determined by plating 100 \u0026micro;L of the control, test cultures, or the respective dilutions at the indicated times onto sheep blood agar plates (ThermoFisher, Spain). Plates were incubated for 24 hours at 37 \u0026ordm;C, and after colony counts, the log\u003csub\u003e10\u003c/sub\u003e of viable cells (CFU/mL) was determined. Bactericidal activity was defined as a reduction of \u0026ge;\u0026thinsp;3 log\u003csub\u003e10\u003c/sub\u003e CFU/mL from the initial inoculum.\u003c/p\u003e\n\u003ch3\u003eCheckerboard assay\u003c/h3\u003e\n\u003cp\u003eThe assay was performed on a 96-well plate in duplicate as previously described\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Colistin, imipenem, ceftazidime or tigecycline were two-fold serially diluted along the x-axis, whereas ENOblock was two-fold serially diluted along the y axis to create a matrix, where each well consists of a combination of both agents at different concentrations. Bacterial cultures grown overnight were then diluted in saline to 0.5 McFarland turbidity, followed by 1:50 further dilution LB and inoculation on each well to achieve a final concentration of approximately 5.5x10\u003csup\u003e5\u003c/sup\u003e CFU/mL. The 96-well plates were then incubated at 37\u0026deg;C for 18 hours and examined for visible turbidity. The fractional inhibitory concentration (FIC) of the colistin, imipenem, ceftazidime or tigecycline was calculated by dividing the MIC of colistin, imipenem, ceftazidime or tigecycline in the presence of ENOblock by the MIC of colistin, imipenem, ceftazidime or tigecycline alone. Similarly, the FIC of ENOblock was calculated by dividing the MIC of ENOblock in the presence of imipenem, ceftazidime or tigecycline alone. The FIC index was the summation of both FIC values. FIC index values of \u0026le;\u0026thinsp;0.5 and \u0026gt;\u0026thinsp;5 were interpreted as synergistic and non-synergistic, respectively.\u003c/p\u003e\n\u003ch3\u003eEIIP/AQVN filter\u003c/h3\u003e\n\u003cp\u003eSpecific recognition and targeting between interacting biological molecules at distances\u0026thinsp;\u0026gt;\u0026thinsp;5 \u0026Aring; were determined by the average quasi-valence number (AQVN) and the Electron-ion interaction potential (EIIP) derived from the general model pseudopotential\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eEIIP\u0026thinsp;=\u0026thinsp;0.25 Z* sin(1.04 π Z*) (1)\u003c/p\u003e \u003cp\u003ewhere Z* is the AQVN determined by:\u003c/p\u003e \u003cp\u003eZ* = \u0026sum;m(ni Zi/N) (2)\u003c/p\u003e \u003cp\u003ewhere Zi is the valence number of the ith atomic component, ni is the number of atoms of the ith component, m is the number of atomic components in the molecule, and N is the total number of atoms. EIIP values are computed using Equations (1) and (2) and are expressed in Rydberg units (Ry).\u003c/p\u003e \u003cp\u003eAQVN and EIIP are unique physical properties that characterize long-range interactions between biological molecules among the 3300 molecular descriptors currently in use\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. It has been shown that the EIIP and AQVN of organic molecules strongly correlate with their biological activity (mutagenicity, carcinogenicity, toxicity, antibiotic and cytostatic activity, etc.)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHuman cell culture\u003c/h2\u003e \u003cp\u003eHeLa cells were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), vancomycin (50 mg/L), gentamicin (20 mg/L), and amphotericin B (0.25 mg/L) (Invitrogen, Spain), and 1% HEPES in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. The HeLa cells were routinely passaged every 3 or 4 days. Immediately before infection, HeLa cells were washed three times with prewarmed PBS and further incubated in DMEM without FBS and antibiotics\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAdhesion and invasion assays\u003c/h3\u003e\n\u003cp\u003eHeLa cells were infected with \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 and Ab CR17 strains at a concentration of 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL, in the absence and presence of 1xMIC of ENOblock at a multiplicity of infection (MOI) of 100. The infection was carried out for two hours with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C in three independent experiments. After that, the infected HeLa cells were washed five times with pre-warmed PBS and lysed with 0.5% Triton X-100. Diluted lysates were plated onto LB agar and incubated at 37\u0026deg;C for 24 hours to enumerate the developed colonies and determine the number of bacteria that had attached to the HeLa cells\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition, to determine the number of colonies that entered inside the HeLa cells, the wells were washed with phosphate buffered saline and incubated for 30 minutes in the presence of DMEM plus gentamicin (256 \u0026micro;g/mL), in order to kill the bacteria present in the area. Then, the wells were washed with phosphate buffered saline to remove gentamicin. The number of colonies that entered inside HeLa cells was determined as described above\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eCellular toxicity of ENOblock\u003c/h3\u003e\n\u003cp\u003eHeLa cells and macrophages differentiated from THP-1 cells\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e were incubated with ENOblock at different concentration ranged from 0.5 to 256 mg/L for 24 hours with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Prior the evaluation of the ENOblock cytotoxicity, HeLa and macrophage cells were washed three times with prewarmed PBS 1X. Subsequently, quantitative cytotoxicity was evaluated by measuring the mitochondrial reduction activity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The percentage of cytotoxicity was calculated from the absorbance at 570 nm as follow: [(Absorbance 570 nm of treated cells/Absorbance 570 nm mean of untreated cells) \u0026times; 100]. The cytotoxic concentration 50% (CC\u003csub\u003e50\u003c/sub\u003e) value was determined using GraphPad Prism 9.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eImmunofluorescence assay was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Briefly, the HeLa cells plated on coverslips were incubated with \u003cem\u003eA. baumannii\u003c/em\u003e CR17 and \u003cem\u003eE. coli\u003c/em\u003e MCR1\u003csup\u003e+\u003c/sup\u003e strains for two hours, and later were incubated with ENOblock (0 and 1xMIC, 30 min) at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37\u0026deg;C. Bacterial cells were removed and HeLa cells were washed five times with cold PBS. HeLa cells on the coverslips were fixed in methanol for 8 min at -20\u0026deg;C, permeabilized with 0.5% Triton X-100 and blocked with 20% pork serum in PBS. Primary antibodies: anti-OmpA of \u003cem\u003eA. baumannii\u003c/em\u003e (ThermoFischer, Spain), mouse anti-\u003cem\u003eE. coli\u003c/em\u003e (Abcam, Spain), and rabbit anti-human fibronectin (Merck, Spain) were used at dilution of 1:25, 1:25 and 1:50, respectively, in PBS containing 1% bovine serum albumin (BSA) for 2 hours. After washing with PBS, the coverslips were incubated with their respective secondary antibodies: Alexa488-conjugated goat anti-mouse IgG, and Alexa594-conjugated goat anti-rabbit IgG (Invitrogen, Spain) at dilution of 1:50, 1:50 and 1:100, respectively, in PBS containing 1% BSA for 1 hours. The fixed coverslips were incubated for 10 minutes at room temperature with DAPI (Applichem, Germany) (0.5 \u0026micro;g/mL), washed with PBS, mounted in fluorescence mounting medium \u0026ldquo;Prolong Diamond Antifade Mountant\u0026rdquo; (Invitrogen, Spain), and visualized using fluorescence microscopy Zeiss Axio Imager 2 (Zeiss, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBacterial cytological profiling\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eFluorescent microscopy\u003c/h2\u003e \u003cp\u003eOvernight cultures of \u003cem\u003eA. baumannii\u003c/em\u003e were diluted 1:100 in LB broth and incubated on a roller at 30\u0026deg;C until the OD600 reached 0.2. ENOblock was added to the bacterial cultures at MIC levels prior to observation under a fluorescence microscope at various time points. After each timepoint, the cultures were stained with 2 \u0026micro;g/mL FM4-64, 2 \u0026micro;g/mL DAPI, and 0.5 \u0026micro;M SYTOX Green. The bacterial cells were then harvested by centrifugation at 6,000 x g for one minute and resuspended in 1/10 of the original volume. A small amount of the concentrated bacterial cultures was placed on an agarose pad (1.2% agarose in 10% LB broth) on concave glass slides for microscopy. Consistent experimental settings and imaging parameters were maintained throughout all experiments included in the statistical analysis of the antibiotic training sets.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImage data analysis.\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe raw images from the fluorescent microscope were preprocessed using ImageJ software\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, and cell features were extracted with CellProfiler 4.0 software\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. After data extraction, the ENOblock-treated cell profiles were analyzed alongside antibiotic-treated cell profiles from previous studies using an analysis pipeline from previous research\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Briefly, the data was transformed using QuantileTransformer\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, and outliers were removed using hierarchical density-based spatial clustering of applications with noise (HDBSCAN)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This analysis utilized a morphological feature set from previous studies\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Finally, the dimension of the data set was reduced and visualized through data clustering using pairwise controlled manifold approximation (PaCMAP)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDocking and molecular modelling\u003c/h2\u003e \u003cp\u003eThe crystal structure of enolase C-terminal of \u003cem\u003eA. baumannii\u003c/em\u003e was predicted by AlphaFold\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. This target protein was modified using Autodock tools 1.5.6 software; including ligand and water removal, hydrogen addition, and incorporation of Kollman charges. The resultant files were saved in pdbqt format.\u003c/p\u003e \u003cp\u003eThe ligand \"ENOblock\" was downloaded from Pubchem (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) in SMILES format (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/compound/24012277\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/compound/24012277\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Openbabel 3.1.1 software was used to create the 3D chemical structure, energy minimization, hydrogen atoms addition and establishment of a neutral pH. Resulting ligand was saved in MOL.2 format. Gasteiger charges computation for the ligand structure was performed using Autodock tools 1.5.6, with the output saved in pdbqt format. Autodock tools 1.5.6 was used for docking and subsequent analysis of the docking results.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of enolase knockout from\u003c/b\u003e \u003cb\u003eA. baumannii\u003c/b\u003e \u003cb\u003eATCC 17978\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo construct an \u003cem\u003eenolase\u003c/em\u003e knockout from \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978, we followed the protocol described previously\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Briefly, an internal \u003cem\u003eenolase\u003c/em\u003e 482-bp fragment obtained by PCR amplification with the primers enolase IntUp and enolase IntLw (Table S1) was cloned into pGEM-T (Promega, Spain) to give plasmid enolase-pGEM-T by using T4 DNA ligase (Promega, Spain). The resulting construct incorporated into \u003cem\u003eE. coli\u003c/em\u003e DH5α was purified and electroporated into ATCC 17978 to knock out the \u003cem\u003eenolase\u003c/em\u003e gene. Transformants were selected on LB agar plates containing 80 \u0026micro;g/mL ticarcillin. The \u003cem\u003eenolase\u003c/em\u003e gene disruption within the resulting strain, designated Ab Δ\u003cem\u003eeno\u003c/em\u003e, was confirmed by PCR using a combination of primers matching the upstream region of \u003cem\u003eenolase\u003c/em\u003e gene and the pGEM-T Easy vector.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBacterial enolase activity assay\u003c/h2\u003e \u003cp\u003eEnolase activity was determined in three independent experiments following the instructions of Enolase Activity Assay kit (Merck, Spain). Briefly, a bacterial inoculum of 10\u003csup\u003e6\u003c/sup\u003e CFU/mL, after centrifugation, was incubated with a master mix composed by enolase substrate, peroxidase substrate, and the necessary converters for the colorimetric reaction at 25 \u0026ordm;C. Enolase enzymatic activity, reported in mU/mL, was measured at OD\u003csub\u003e570 nm\u003c/sub\u003e every two-three minutes for one hour using a microtiter plate reader (Tecan Spark, Austria) and calculated using the specific formula: ΔA570 = (A570)\u003csub\u003efinal\u003c/sub\u003e - (A570)\u003csub\u003einitial\u003c/sub\u003e,\u003c/p\u003e \u003cp\u003eand the following equation:\u003c/p\u003e \u003cp\u003eenolase Activity = (B \u0026times; Sample Dilution Factor) / (Reaction Time \u0026times; V)\u003c/p\u003e \u003cp\u003ewhere: \u003cb\u003eB\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Amount (nmol) of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e generated between T\u003csub\u003einitial\u003c/sub\u003e and T\u003csub\u003efinal\u003c/sub\u003e,\u003c/p\u003e \u003cp\u003e \u003cb\u003eReaction Time\u003c/b\u003e\u0026thinsp;=\u0026thinsp;T\u003csub\u003efina\u003c/sub\u003el \u0026ndash; T\u003csub\u003einitial\u003c/sub\u003e (minutes), and\u003c/p\u003e \u003cp\u003e \u003cb\u003eV\u003c/b\u003e\u0026thinsp;=\u0026thinsp;volume of sample (mL) added to the well\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBacterial growth curves\u003c/h2\u003e \u003cp\u003eTo determine the antibacterial of ENOblock against \u003cem\u003ewild-type\u003c/em\u003e and enolase-defieicent \u003cem\u003eA. baumannii\u003c/em\u003e, duplicate bacterial growth curves of the \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 and its isogenic deficient in enolase (Ab Δ\u003cem\u003eeno\u003c/em\u003e) strain were performed in duplicate in 96-well plate (Deltlab, Spain). An initial inoculum of 5x10\u003csup\u003e5\u003c/sup\u003e CFU/mL was prepared in LB in the presence of 1x, 2x and 4x MIC of ENOblock. A drug-free broth was evaluated in parallel as a control. Plates were incubated at 37\u0026deg;C with shaking, and bacterial growth was monitored for 24 hours using a microtiter plate reader (Tecan Spark, Austria).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGalleria mellonella\u003c/b\u003e \u003cb\u003einfection model\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eG. mellonella\u003c/em\u003e infection model with ATCC 17978 strain was established by haemocoel bacterial inoculation. Briefly, caterpillars obtained from Artroposfera (Toledo, Spain) were inoculated with 10 \u0026micro;L of the bacterial suspensions, which were incubated for 20\u0026ndash;24 hours in LB at 37\u0026deg;C. The minimal bacterial lethal dose 100 (MLD100) and LD50 were determined by inoculating various groups of larvae (8 \u003cem\u003eG. mellonella\u003c/em\u003e per group) with decreasing amounts of ATCC 17978 strains inocula from 10\u003csup\u003e6\u003c/sup\u003e to 10\u003csup\u003e2\u003c/sup\u003e CFU/mL, and monitoring the survival of the larvae for 7 days.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTherapeutic efficacy of ENOblock in\u003c/b\u003e \u003cb\u003eG. mellonella\u003c/b\u003e \u003cb\u003einfection model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe efficacy of the ENOblock treatment was tested in \u003cem\u003eG. mellonella\u003c/em\u003e survival assay as previously described\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Caterpillars were injected by the 10 \u0026micro;L of suspension containing MLD of ATCC 17978. Treatment with 4xMIC of ENOblock was injected one hour post-infection. A group pf larvae injected with 10 \u0026micro;L of sterile PBS was included as control. After inoculation, the larvae were incubated at 37\u0026deg;C in the dark and death was assessed over 3 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eGroup data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the means (SEM). The student \u003cem\u003et\u003c/em\u003e-test was used to determine differences between means using the GraphPad Prism 9 (version 9.3.1; GraphPad Software, LLC.). For the \u003cem\u003eG. mellonella\u003c/em\u003e survival model, a Kaplan-Meier test was performed to determine the difference between mortality rates. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHigh-throughput screening for repurposing drugs as antimicrobial agents\u003c/h2\u003e \u003cp\u003eWe developed and validated a high-throughput screen assay using the \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 and \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 strains, as well as their respective MDR strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In total, we screened 2,464 compounds from the EU-OPENSCREEN ECBL Pilot library. We identified 33 compounds (1.32% of the total compounds) with inhibitory activity of \u0026ge;\u0026thinsp;70% against at least one or both MDR strains of \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. Of these 33 compounds, 7 showed MICs of \u0026le;\u0026thinsp;100 \u0026micro;M against the reference and MDR strains of \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Among these 7 compounds, ENOblock (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) was chosen for further studies. This compound was active against the \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 and \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 strains, with AC\u003csub\u003e50\u003c/sub\u003e values of 23.57 \u0026micro;M and 46.86 \u0026micro;M, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eENOblock is active against\u003c/b\u003e \u003cb\u003eA. baumannii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo confirm the activity of ENOblock against clinical isolates of \u003cem\u003eA. baumannii\u003c/em\u003e, ENOblock was tested against 14 and 18 colistin-resistant and carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e isolates. The results of the MICs tests are displayed in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The MICs ranged from 8 to 32 mg/L and 16 to 32 mg/L for ENOblock against colistin and carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e, respectively. The reference strain ATCC 17978 present an ENOblock MIC of 8 mg/L. The MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e concentrations, which represent the concentration effective for 50 and 90% of the isolates tested, respectively, for ENOblock against colistin and carbapenems-resistant isolates were 16 and 32 mg/L, respectively. However, the MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e for colistin were 256 and \u0026gt;\u0026thinsp;256 mg/L, and for carbapenems were 16 and 64 mg/l (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of note, the ENOblock MIC\u003csub\u003e90\u003c/sub\u003e is three times below the CC\u003csub\u003e50\u003c/sub\u003e of ENOblock in HeLa and macrophage cells (data not shown).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibacterial activity of ENOblock in colistin-resistant \u003cem\u003eA. baumannii\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColistin MIC (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eENOblock MIC (mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#99\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#113\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAb CR17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e90\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibacterial activity of ENOblock in carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eImipenem/meropenem\u003c/p\u003e \u003cp\u003eMIC (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eENOblock\u003c/p\u003e \u003cp\u003eMIC (mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#37\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#40\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#53\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#286\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#288\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#289\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#295\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#298\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#299\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#405\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#410\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#414\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#416\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#417\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#440\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#441\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e#448\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e90\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eUsing time-course assays, we evaluated the bactericidal activity of ENOblock against ATCC 17978 and Ab CR17 strains. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA illustrates that ENOblock (2x and 4xMIC for ATCC 17978 strain) exhibited bactericidal effect after 2, 4 and 8 hours, reducing the bacterial count by over 3 log\u003csub\u003e10\u003c/sub\u003e CFU/mL compared to 0 hours. For the Ab CR17 strain, ENOblock (1x, 2x and 4xMIC for Ab CR17 strain) demonstrated a bactericidal effect after 2, 4 and 8 hours by reducing the bacterial count by over 3 log\u003csub\u003e10\u003c/sub\u003e CFU/mL, compared to 0 hours.\u003c/p\u003e\u003cp\u003eIt is well known that the development of new repurposed drugs includes the assessment of the presence of synergy with clinically used antibiotics. To this end, we conducted a virtual screening of ENOblock in combination with different antibiotics (colistin, imipenem, ceftazidime and tigecycline) using the EIIP/AQVN criterion to overcome bacterial resistance (Table S2). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB suggests that colistin and ENOblock with similar electronic properties, as indicated by their EIIP and AQVN values, tend to exhibit synergistic effects. Conversely, no synergistic activity was observed between the rest of antibiotics and ENOblock with different electronic properties. To confirm experimentally the \u003cem\u003ein silico\u003c/em\u003e synergistic effect of ENOblock with colistin against \u003cem\u003eA. baumannii\u003c/em\u003e, checkerboard assay was performed. This assay indicated that ENOblock had a synergistic effect with colistin by enhancing the activity of colistin against ATCC 17978 and Ab CR17, resulting in an FIC index (FICI) of \u0026le;\u0026thinsp;0.5. In contrast, the combination of ENOblock with other antibiotics such as imipenem, ceftazidime and tigecycline did not increase their activities, yielding a FICI\u0026thinsp;\u0026gt;\u0026thinsp;0.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eENOblock affects the\u003c/b\u003e \u003cb\u003eA. baumannii\u003c/b\u003e\u003cb\u003e-host interaction\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the effect of ENOblock on the interaction between \u003cem\u003eA. baumannii\u003c/em\u003e and host cells, we studied the adherence and invasion of the ATCC 17978 and Ab CR17 strains on HeLa cells for two hours in the presence of ENOblock. We found that treatment with ENOblock at 1xMIC reduced the counts of adherent ATCC 17978 and Ab CR17 strains on HeLa cells by 47% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 31% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), respectively. Notably, a more significant reduction was observed in the invasion of both strains, with ENOblock treatment reducing invasive counts by 76% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 46% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, immunostaining of infected HeLa cells with the ATCC 17978 and Ab CR17 strains, pretreated with ENOblock, showed a significant reduction in \u003cem\u003eA. baumannii\u003c/em\u003e attachment to HeLa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e \u003cb\u003eENOblock inhibits the growth of\u003c/b\u003e \u003cb\u003eA. baumannii\u003c/b\u003e \u003cb\u003evia distinct mechanism of action\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe employed the fluorescence microscopy-based BCP technique, as previously applied to \u003cem\u003eA. baumannii\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, to investigate the mechanism of action of ENOblock against ATCC 17978 strain. The morphological changes induced by ENOblock were compared with those of antibiotics targeting major cellular pathways, including ciprofloxacin CIP (DNA replication), rifampicin RIF (RNA transcription), minocycline MIN (protein translation), piperacillin PIP and meropenem MER (cell wall synthesis), and colistin CST (membrane integrity). BCP results showed that ENOblock-treated cells exhibit unique morphological changes compared to those of antibiotic controls. In particular, membrane blebs were observed, as was the high SYTOX green signal of ENOblock-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), indicating the loss of membrane integrity. However, the ENOblock-treated cells showed different morphological changes from CST-treated cells, a membrane integrity control, indicating that ENOblock interfere with bacterial membrane integrity but possibly in a manner different from CST. Consistent with these differences, the image analysis profiles of ENOblock-treated cells clustered separately from those of untreated, other control antibiotics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). It is conceivable that ENOblock inhibits pathways that are distinct from those targeted by the comparator antibiotics, which collectively represent the most common modes of antibacterial action, in a manner similar to previous studies that have observed distinct profiles\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Altogether, the results showed that the ENOblock profile is distinct from the six antibiotic profiles.\u003c/p\u003e \u003cp\u003e \u003cb\u003eENOblock exhibits rapid permeabilization activity against\u003c/b\u003e \u003cb\u003eA. baumannii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe observation of a higher proportion of ENOblock-treated cells displaying high SYTOX Green intensity prompts us to consider the possibility of observing this impact at earlier time intervals, as membrane permeabilization often happens swiftly within minutes. Prior studies utilizing BCP demonstrated that a compound with the ability to disrupt membranes could impact the bacterial membrane in a mere 10 minutes timeframe\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Consequently, we conducted a temporal examination of SYTOX green staining on cells treated with ENOblock for 10, 30, and 60 minutes. The findings indicated that cells treated with ENOblock exhibited a notable rise in SYTOX intensity just 10 minutes after treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F), in comparison to the control group that did not receive treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Although the fluorescence levels continued to increase after 30 minutes, this rise was not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), indicating that the cells reached a saturation point with SYTOX Green after 10 minutes. Hence, the results indicates that ENOblock rapidly compromises the rigidity of the bacterial cell envelope, potentially resulting in cell death.\u003c/p\u003e \u003cp\u003e \u003cb\u003eENOblock acts on\u003c/b\u003e \u003cb\u003eA. baumannii\u003c/b\u003e \u003cb\u003ethrough the inhibition of enolase\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to shed light on the ENOblock mechanism of action, we docked ENOblock in the C-terminal domain of \u003cem\u003eA. baumannii\u003c/em\u003e enolase. ENOblock exhibited a high docking score. The most stable pose shows that ENOblock binds to Ser371 and Asp207 amine acids through 3 hydrogen bonds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm that enolase is the potential target of ENOblock, we generated an enolase-deficient mutant. Deletion of the \u003cem\u003eenolase\u003c/em\u003e gene in the ATCC17978 strain (Δ\u003cem\u003eeno\u003c/em\u003e) first abolished completely the enolase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and subsequently increased the ENOblock MIC from 8 to 32 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, we examined the antibacterial activity of ENOblock against the ATCC 17978 and Δ\u003cem\u003eeno\u003c/em\u003e strains. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD reveals that the ATCC 17978 strain exhibits rapid growth, reaching an OD of 1 within the first 4 hours. However, a significant disparity in growth is observed between the untreated cells and the cells treated with ENOblock, particularly at higher compound concentrations (16 and 32 mg/L). A different trend of growth inhibition is observed in the Δ\u003cem\u003eeno\u003c/em\u003e strain, which shows a higher OD value compared with \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 strain in the presence of ENOblock treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). This difference in growth can be attributed to the resistance of the mutant strain to the ENOblock, as the absence of enolase may hinder the compound\u0026rsquo;s ability to exert its effect, as indicated by the findings of the molecular docking study.\u003c/p\u003e \u003cp\u003e \u003cb\u003eENOblock presents therapeutic efficacy\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo confirm the \u003cem\u003ein vitro\u003c/em\u003e effect of ENOblock in monotherapy and in combination with colistin against \u003cem\u003eA. baumannii\u003c/em\u003e, and to study this efficacy in a complete organism, we moved to an invertebrate model of infection by \u003cem\u003eA. baumannii\u003c/em\u003e. First, we determined the virulence of the ATCC 17078 strain after haemocoel administration in \u003cem\u003eG. mellonella\u003c/em\u003e. The mortality rates of animals were inoculum concentration dependent. LD\u003csub\u003e50\u003c/sub\u003e and MLD\u003csub\u003e100\u003c/sub\u003e for the ATCC 17978 strain were 10\u003csup\u003e2\u003c/sup\u003e and 1x10\u003csup\u003e5\u003c/sup\u003e CFU/mL, respectively. Subsequently, in a \u003cem\u003eG. mellonella\u003c/em\u003e model of infection, we administered ENOblock (32 mg/L) to animals after haemoceol administration of an MLD\u003csub\u003e100\u003c/sub\u003e of the ATCC 17978 strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Animals receiving treatment with ENOblock showed significantly a greater increase in survival compared with untreated animals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe emergence of MDR \u003cem\u003eA. baumannii\u003c/em\u003e has led to the use of colistin as a last resort for treating severe infections caused by this pathogen. Although colistin resistance is still uncommon, its emergence and spread are regarded as concerns to world health\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDue to the antibacterial effects of various anticancer drugs on \u003cem\u003eA. baumannii\u003c/em\u003e\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, we hypothesized that ENOblock, identified in this study after a HTS of the EU-OPENSCREEN library, might exhibit strong antibacterial activity against colistin- or carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e. After initial antimicrobial confirmation of ENOblock, we additionally tested its susceptibility against 32 clinical isolates of \u003cem\u003eA. baumannii\u003c/em\u003e resistant to colistin or carbapenems (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The ENOblock MIC\u003csub\u003e90\u003c/sub\u003e is 32 mg/L, which is two to more than six times lower than the MIC\u003csub\u003e90\u003c/sub\u003e of carbapenem and colistin, respectively. This MIC\u003csub\u003e90\u003c/sub\u003e value falls within the range of other known antibiotics such as amikacin, amoxicillin-clavulanic acid, ceftazidime-avibactam, and fosfomycin, among others\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is noteworthy that ENOblock demonstrated similar activity against MDR \u003cem\u003eE. coli\u003c/em\u003e (data not shown), consistent with previously published data on the antibacterial activity of the anticancer drug family tamoxifen and its metabolites used in earlier studies\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSpecific AQVN/EIIP domains, combined with structural properties, can serve as effective filters for the virtual screening of molecular libraries to identify new drug candidates, including new antibiotics. Using molecular descriptors, the EIIP and AQVN we have proposed suitable antibiotics for treating MDR bacterial infections\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In this study, we analyzed the electronic properties of ENOblock and antibiotics to which \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 is sensitive. Our analysis suggests that antimicrobials with similar electronic properties tend to act synergistically. However, the limited number of molecules analyzed restricts our ability to establish a criterion for predicting synergy in \u003cem\u003eA. baumannii\u003c/em\u003e. Moreover, studies show that the molecular mechanisms underlying such synergistic effects remain not fully understood\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Nonetheless, the presented results with the observed tendency for ENOblock and colistin, to exhibit synergy suggest that similar electronic properties may contribute to effective antibacterial combinations.\u003c/p\u003e \u003cp\u003eSmall molecules with similar AQVN and EIIP values have previously been shown to interact with the common therapeutic target\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe antibacterial activity of ENOblock at 1x, 2x, and 4xMIC against the colistin-resistant strain (Ab CR17) is higher than against the colistin-susceptible reference strain (ATCC 17978). This result may be related to differences in the cell wall structure of the two strains, where colistin-resistant strains of \u003cem\u003eA. baumannii\u003c/em\u003e are more permeable than colistin-susceptible strains\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe antimicrobial activity of ENOblock identified in this study suggests promising potential that warrants further exploration \u003cem\u003ein vivo\u003c/em\u003e after determining its pharmacokinetic parameters. However, \u003cem\u003ein vitro\u003c/em\u003e bacterial growth showed a progressive regrowth of the ATCC 17978 strain after treatment with ENOblock at 1xMIC, suggesting that this strain may have acquired resistance to this compound. It is worth noting that the MIC of ENOblock against the ATCC 17978 strain in these time-kill assay conditions is 8 mg/L, which is below the 2x and 4xMIC of ENOblock. Further investigations, including the determination of its concentration during the time-kill assay, are necessary to better understand the regrowth of this strain in the presence of ENOblock.\u003c/p\u003e \u003cp\u003eAdditionally, BCP analysis showed that ENOblock-treated \u003cem\u003eA. baumannii\u003c/em\u003e exhibits distinct morphological changes compared to comparator antibiotics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D), suggesting that ENOblock inhibits pathways different from those targeted by the comparator antibiotics. Membrane-disruptive agents can be classified into various subcategories, each exhibiting a unique structure on BCP. Nevertheless, the BCP profile of ENOblock-treated cells observed in this study did not precisely correspond to any previously described profiles\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Consequently, the specific target on the membrane for ENOblock could not be determined, but the presence of SYTOX Green indicates that ENOblock can permeabilize membranes. Therefore, future studies should investigate the precise locations on the bacterial membrane where ENOblock is active.\u003c/p\u003e \u003cp\u003eIt is widely known that ENOblock inhibits enolase activity in eukaryotic cells\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, and enolase, a cytoplasmic glycolytic enzyme, is a key component of the RNA degradosome in Gram-negative bacteria such as \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. For this reason, we decided to conduct a preliminary computational study to determine if ENOblock could potentially bind to enolase in \u003cem\u003eA. baumannii\u003c/em\u003e. As a result, ENOblock exhibited a better docking score. Deletion of \u003cem\u003eenolase\u003c/em\u003e gene in \u003cem\u003eA. baumannii\u003c/em\u003e increased the ENOblock MIC four-fold and increased bacterial growth in the presence of ENOblock, suggesting that enolase could be a potential target of ENOblock in \u003cem\u003eA. baumannii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eImportantly, two studies have reported that enolase also resides on the cell wall outer membrane of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e and mediates the binding of \u003cem\u003eP. aeruginosa\u003c/em\u003e to plasminogen on host cells\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. In this study, we showed that ENOblock reduces the interaction of \u003cem\u003eA. baumannii\u003c/em\u003e with host cells, possibly by affecting the plasminogen-binding activity of \u003cem\u003eA. baumannii\u003c/em\u003e outer membrane enolase\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Consequently, the possibility of multi-targeting, with ENOblock binding to both cytosolic and membranal enolase, might contribute to its overall activity, which is an attractive aspect of ENOblock's antibacterial properties.\u003c/p\u003e \u003cp\u003eWe have demonstrated through various assays that ENOblock can be repurposed as an antibacterial agent. However, the ultimate goal of this compound is to achieve good therapeutic efficacy in animal models of infection. So far, ENOblock at 32 mg/L has been able to increase animal survival in presence of \u003cem\u003eA. baumannii\u003c/em\u003e. This result is consistent with previous observations that deletion of \u003cem\u003eenolase\u003c/em\u003e gene abolishes the virulence of \u003cem\u003eP. aeruginosa\u003c/em\u003e in a murine acute pneumonia model\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, this drug discovery approach ought to be viewed as the first step in creating a brand-new class of antimicrobial drugs. The efficacy of ENOblock can be investigated further by combining this newly repurposed compound with clinically used antibiotics (like colistin) in \u003cem\u003ein vivo\u003c/em\u003e experiments, with the aim of reducing mortality and improving therapeutic efficacy in cases of severe infections, even with the current antimicrobial therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eI.M.P., A.M.R., M.C., P.S., T.S., V.V. \u0026amp; A.H. methodology, investigation, formal analysis. A.P.P, S.G., O.G. A.H. \u0026amp; P.N. writing-review and editing, methodology, investigation. Y.S. writing-review and editing, supervision, funding acquisition, conceptualization.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis research was funded by the Ministerio de Ciencia e Innovaci\u0026oacute;n, Agencia Estatal de Investigaci\u0026oacute;n, Fondo Europeo de Desarrollo Regional, MCIN/AEI/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.13039/501100011033/FEDER\u003c/span\u003e\u003cspan address=\"10.13039/501100011033/FEDER\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, UE (Grant PID2022-136357OBI00), and (Grant CEX2020-001088-M-20-5), by the Consejer\u0026iacute;a de Universidad, Investigaci\u0026oacute;n e Innovaci\u0026oacute;n de la Junta de Andaluc\u0026iacute;a (Grant ProyExcel_00116), by the National Research Council of Thailand (NRCT) and Mahidol University: N42A650368, and by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Grant number 451-03-66/2024-03/200017). We acknowledge EU-OPENSCREEN ERIC for providing its compound collection and Fundaci\u0026oacute;n MEDINA HTS antimicrobial screening platform to support the discovery of the antibacterial activity of the compound described in the presented work. This article is based upon work from COST Action EURESTOP, CA21145, supported by COST (European Cooperation in Science and Technology). A.M.R is supported by a doctoral fellowship PRE2022-104318, from the Agencia Estatal de Investigaci\u0026oacute;n, Ministerio de Ciencia e Innovaci\u0026oacute;n.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMorris S, Cerceo E (2020) Trends, epidemiology, and management of multi-drug resistant gram-negative bacterial infections in the hospitalized setting. 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Front Microbiol 7:1999\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ENOblock, enolase, treatment, bacteria, infection, Acinetobacter baumannii","lastPublishedDoi":"10.21203/rs.3.rs-5059044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5059044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHigh-throughput screening studies provide an additional approach to discovering repurposed drugs for antimicrobial treatments. In this work, we report the identification of ENOblock, an anticancer drug, as a novel antibiotic class. We computationally and experimentally validated that ENOblock synergizes with the last resort antibiotic, the colistin. Additionally, we identified enolase as the potential bacterial target for ENOblock. The \u003cem\u003ein silico\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e antibacterial activity of ENOblock translated into potent \u003cem\u003ein vivo\u003c/em\u003e efficacy in animal infection models. Collectively, the preclinical data support the selection of ENOblock as a promising candidate for antimicrobial development, with the potential to address the urgent threat of infections caused by \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"A novel antibiotic class targeting the enolase of Acinetobacter baumannii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-06 09:22:58","doi":"10.21203/rs.3.rs-5059044/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5ec6d45e-fe5f-484a-b2d4-cbcd5ec4ed26","owner":[],"postedDate":"January 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":37570842,"name":"Biological sciences/Microbiology/Antimicrobials"},{"id":37570843,"name":"Health sciences/Pathogenesis/Infection"}],"tags":[],"updatedAt":"2025-04-26T19:35:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-06 09:22:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5059044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5059044","identity":"rs-5059044","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}