Characterization and Abundance of Plasmid-Dependent Alphatectivirus Bacteriophages.

Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis.

A large chromosomal inversion affects antimicrobial sensitivity of Escherichia coli to sodium deoxycholate.

Resistance to antimicrobials is normally caused by mutations in the drug targets or genes involved in antimicrobial activation or expulsion. Here we show that an Escherichia coli strain, named DOC14, selected for increased resistance to the bile salt sodium deoxycholate, has no mutations in any ORF, but instead has a 2.1 Mb chromosomal inversion. The breakpoints of the inversion are two inverted copies of an IS5 element. Besides lowering deoxycholate susceptibility, the IS5-mediated chromosomal inversion in the DOC14 mutant was found to increase bacterial survival upon exposure to ampicillin and vancomycin, and sensitize the cell to ciprofloxacin and meropenem, but does not affect bacterial growth or cell morphology in a rich medium in the absence of antibacterial molecules. Overall, our findings support the notion that a large chromosomal inversion can benefit bacterial cells under certain conditions, contributing to genetic variability available for selection during evolution. The DOC14 mutant paired with its isogenic parental strain form a useful model as bacterial ancestors in evolution experiments to study how a large chromosomal inversion influences the evolutionary trajectory in response to various environmental stressors.

Human metapneumovirus activates NOD-like receptor protein 3 inflammasome via its small hydrophobic protein which plays a detrimental role during infection in mice.

NOD-like receptor protein 3 (NLRP3) inflammasome activation triggers caspase-1 activation-induced maturation of interleukin (IL)-1ß and IL-18 and therefore is important for the development of the host defense against various RNA viral diseases. However, the implication of this protein complex in human metapneumovirus (HMPV) disease has not been fully studied. Herein, we report that NLRP3 inflammasome plays a detrimental role during HMPV infection because NLRP3 inflammasome inhibition protected mice from mortality and reduced weight loss and inflammation without impacting viral replication. We also demonstrate that NLRP3 inflammasome exerts its deleterious effect via IL-1ß production since we observed reduced mortality, weight loss and inflammation in IL-1ß-deficient (IL-1ß-/-) mice, as compared to wild-type animals during HMPV infection. Moreover, the effect on these evaluated parameters was not different in IL-1ß-/- and wild-type mice treated with an NLRP3 inflammasome inhibitor. The production of IL-1ß was also abrogated in bone marrow derived macrophages deficient for NLRP3. Finally, we show that small hydrophobic protein-deleted recombinant HMPV (HMPV ΔSH) failed to activate caspase-1, which is responsible for IL-1ß cleavage and maturation. Furthermore, HMPV ΔSH-infected mice had less weight loss, showed no mortality and reduced inflammation, as compared to wild-type HMPV-infected mice. Thus, NLRP3 inflammasome activation seems to be triggered by HMPV SH protein in HMPV disease. In summary, once activated by the HMPV SH protein, NLRP3 inflammasome promotes the maturation of IL-1ß, which exacerbates HMPV-induced inflammation. Therefore, the blockade of IL-1ß production by using NLRP3 inflammasome inhibitors might be a novel potential strategy for the therapy and prevention of HMPV infection.

Deep in the Bowel: Highly Interpretable Neural Encoder-Decoder Networks Predict Gut Metabolites from Gut Microbiome.

BACKGROUND: Technological advances in next-generation sequencing (NGS) and chromatographic assays [e.g., liquid chromatography mass spectrometry (LC-MS)] have made it possible to identify thousands of microbe and metabolite species, and to measure their relative abundance. In this paper, we propose a sparse neural encoder-decoder network to predict metabolite abundances from microbe abundances. RESULTS: Using paired data from a cohort of inflammatory bowel disease (IBD) patients, we show that our neural encoder-decoder model outperforms linear univariate and multivariate methods in terms of accuracy, sparsity, and stability. Importantly, we show that our neural encoder-decoder model is not simply a black box designed to maximize predictive accuracy. Rather, the network's hidden layer (i.e., the latent space, comprised only of sparsely weighted microbe counts) actually captures key microbe-metabolite relationships that are themselves clinically meaningful. Although this hidden layer is learned without any knowledge of the patient's diagnosis, we show that the learned latent features are structured in a way that predicts IBD and treatment status with high accuracy. CONCLUSIONS: By imposing a non-negative weights constraint, the network becomes a directed graph where each downstream node is interpretable as the additive combination of the upstream nodes. Here, the middle layer comprises distinct microbe-metabolite axes that relate key microbial biomarkers with metabolite biomarkers. By pre-processing the microbiome and metabolome data using compositional data analysis methods, we ensure that our proposed multi-omics workflow will generalize to any pair of -omics data. To the best of our knowledge, this work is the first application of neural encoder-decoders for the interpretable integration of multi-omics biological data.

In vitro synergy between sodium deoxycholate and furazolidone against enterobacteria.

BACKGROUND: Antimicrobial combinations have been proven as a promising approach in the confrontation with multi-drug resistant bacterial pathogens. In the present study, we identify and characterize a synergistic interaction of broad-spectrum nitroreductase-activated prodrugs 5-nitrofurans, with a secondary bile salt, Sodium Deoxycholate (DOC) in growth inhibition and killing of enterobacteria. RESULTS: Using checkerboard assay, we show that combination of nitrofuran furazolidone (FZ) and DOC generates a profound synergistic effect on growth inhibition in several enterobacterial species including Escherichia coli, Salmonella enterica, Citrobacter gillenii and Klebsiella pneumoniae. The Fractional Inhibitory Concentration Index (FICI) for DOC-FZ synergy ranges from 0.125 to 0.35 that remains unchanged in an ampicillin-resistant E. coli strain containing a ß-lactamase-producing plasmid. Findings from the time-kill assay further highlight the synergy with respect to bacterial killing in E. coli and Salmonella. We further characterize the mechanism of synergy in E. coli K12, showing that disruption of the tolC or acrA genes that encode components of multidrug efflux pumps causes, respectively, a complete or partial loss, of the DOC-FZ synergy. This finding indicates the key role of TolC-associated efflux pumps in the DOC-FZ synergy. Overexpression of Nitric Oxide-detoxifying enzyme Hmp results in a three-fold increase in FICI for DOC-FZ interaction, suggesting a role of nitric oxide in the synergy. We further demonstrate that DOC-FZ synergy is largely independent of NfsA and NfsB, the two major activation enzymes of the nitrofuran prodrugs. CONCLUSIONS: This study is to our knowledge the first report of nitrofuran-deoxycholate synergy against Gram-negative bacteria, offering potential applications in antimicrobial therapeutics. The mechanism of DOC-FZ synergy involves FZ-mediated inhibition of TolC-associated efflux pumps that normally remove DOC from bacterial cells. One possible route contributing to that effect is via FZ-mediated nitric oxide production.

Novel 5-Nitrofuran-Activating Reductase in Escherichia coli.

The global spread of multidrug-resistant enterobacteria warrants new strategies to combat these pathogens. One possible approach is the reconsideration of "old" antimicrobials, which remain effective after decades of use. Synthetic 5-nitrofurans such as furazolidone, nitrofurantoin, and nitrofurazone are such a class of antimicrobial drugs. Recent epidemiological data showed a very low prevalence of resistance to this antimicrobial class among clinical Escherichia coli isolates in various parts of the world, forecasting the increasing importance of its uses to battle antibiotic-resistant enterobacteria. However, although they have had a long history of clinical use, a detailed understanding of the 5-nitrofurans' mechanisms of action remains limited. Nitrofurans are known as prodrugs that are activated in E. coli by reduction catalyzed by two redundant nitroreductases, NfsA and NfsB. Furazolidone, nevertheless, retains relatively significant antibacterial activity in the nitroreductase-deficient ΔnfsA ΔnfsBE. coli strain, indicating the presence of additional activating enzymes and/or antibacterial activity of the unreduced form. Using genome sequencing, genetic, biochemical, and bioinformatic approaches, we discovered a novel 5-nitrofuran-activating enzyme, AhpF, in E. coli The discovery of a new nitrofuran-reducing enzyme opens new avenues for overcoming 5-nitrofuran resistance, such as designing nitrofuran analogues with higher affinity for AhpF or screening for adjuvants that enhance AhpF expression.

Using the IPTG-Inducible Pgrac212 Promoter for Overexpression of Human Rhinovirus 3C Protease Fusions in the Cytoplasm of Bacillus subtilis Cells.

Expression and secretion of recombinant proteins in the endotoxin-free bacterium, Bacillus subtilis, has been thoroughly studied, but overexpression in the cytoplasm has been limited to only a few proteins. Here, we used the robust IPTG-inducible promoter, Pgrac212, to overexpress human rhinovirus 3C protease (HRV3C) in the cytoplasm of B. subtilis cells. A novel solubility tag, the N-terminal domain of the lysS gene of B. subtilis coding for a lysyl-tRNA synthetase was placed at the N terminus with a cleavage site for the endoprotease HRV3C, followed by His-HRV3C or His-GST-HRV3C. The recombinant protease was purified by using a Ni-NTA column. In this study, the His-HRV3C and His-GST-HRV3C proteases were overexpressed in the cytoplasm of B. subtilis at 11% and 16% of the total cellular proteins, respectively. The specific protease activities were 8065 U/mg for His-HRV3C and 3623 U/mg for His-GST-HRV3C. The purified enzymes were used to cleave two different substrates followed by purification of the two different protein targets, the green fluorescent protein and the beta-galactosidase. In conclusion, the combination of an inducible promoter Pgrac212 and a solubility tag allowed the overexpression of the HRV3C protease in the cytoplasm of B. subtilis. The resulting fusion protein was purified using a nickel column and was active in cleaving target proteins to remove the fusion tags. This study offers an effective method for producing recombinant proteins in the cytoplasm of endotoxin-free bacteria.

Platelet activation and aggregation promote lung inflammation and influenza virus pathogenesis.

RATIONALE: The hallmark of severe influenza virus infection is excessive inflammation of the lungs. Platelets are activated during influenza, but their role in influenza virus pathogenesis and inflammatory responses is unknown. OBJECTIVES: To determine the role of platelets during influenza A virus infections and propose new therapeutics against influenza. METHODS: We used targeted gene deletion approaches and pharmacologic interventions to investigate the role of platelets during influenza virus infection in mice. MEASUREMENTS AND MAIN RESULTS: Lungs of infected mice were massively infiltrated by aggregates of activated platelets. Platelet activation promoted influenza A virus pathogenesis. Activating protease-activated receptor 4, a platelet receptor for thrombin that is crucial for platelet activation, exacerbated influenza-induced acute lung injury and death. In contrast, deficiency in the major platelet receptor glycoprotein IIIa protected mice from death caused by influenza viruses, and treating the mice with a specific glycoprotein IIb/IIIa antagonist, eptifibatide, had the same effect. Interestingly, mice treated with other antiplatelet compounds (antagonists of protease-activated receptor 4, MRS 2179, and clopidogrel) were also protected from severe lung injury and lethal infections induced by several influenza strains. CONCLUSIONS: The intricate relationship between hemostasis and inflammation has major consequences in influenza virus pathogenesis, and antiplatelet drugs might be explored to develop new antiinflammatory treatment against influenza virus infections.

Annexin V incorporated into influenza virus particles inhibits gamma interferon signaling and promotes viral replication.

UNLABELLED: During the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. We demonstrate here that the expression of annexin V (A5) is upregulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, gamma interferon (IFN-γ)-induced Stat phosphorylation and IFN-γ-induced 10-kDa protein (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN-γ signaling pathway was A5 dependent since downregulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN-γ antiviral immune response by incorporating A5 into their envelope during the budding process. IMPORTANCE: Many enveloped viruses, including influenza A viruses, bud from the plasma membrane of their host cells and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. We demonstrate here that the host protein annexin V is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin V counteracted the antiviral activity of gamma interferon in vitro and in vivo. Thus, these results showed that annexin V incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes.

Plasminogen controls inflammation and pathogenesis of influenza virus infections via fibrinolysis.

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible for the observed effect. However, pharmacological depletion of fibrinogen, the main target of plasminogen reversed disease resistance of plasminogen-deficient mice or mice treated with an inhibitor of plasminogen-mediated fibrinolysis. Therefore, plasminogen contributes to the deleterious inflammation of the lungs and local fibrin clot formation may be implicated in host defense against influenza virus infections. Our studies suggest that the hemostatic system might be explored for novel treatments against influenza.

Switch from protective to adverse inflammation during influenza: viral determinants and hemostasis are caught as culprits.

Influenza viruses cause acute respiratory infections, which are highly contagious and occur as seasonal epidemic and sporadic pandemic outbreaks. Innate immune response is activated shortly after infection with influenza A viruses (IAV), affording effective protection of the host. However, this response should be tightly regulated, as insufficient inflammation may result in virus escape from immunosurveillance. In contrast, excessive inflammation may result in bystander lung tissue damage, loss of respiratory capacity, and deterioration of the clinical outcome of IAV infections. In this review, we give a comprehensive overview of the innate immune response to IAV infection and summarize the most important findings on how the host can inappropriately respond to influenza.

When less is more: shortening the Lpp protein leads to increased vancomycin resistance in Escherichia coli.

Vancomycin is a naturally occurring cell-wall-targeting glycopeptide antibiotic. Due to the low potency of this antibiotic against Gram-negative pathogens, such as Escherichia coli, there is a limited knowledge about interactions between vancomycin and this group of bacteria. Here, we show that an in-frame 63 bp deletion of the lpp gene caused a fourfold increase in vancomycin resistance in E. coli. The resulting protein, LppΔ21, is 21 amino acids shorter than the wild-type Lpp, a helical structural lipoprotein that controls the width of the periplasmic space through its length. The mutant remains susceptible to synergistic growth inhibition by combination of furazolidone and vancomycin; with furazolidone decreasing the vancomycin MIC by eightfold. These findings have clinical relevance, given that the vancomycin concentration required to select the lpp mutation is reachable during typical vancomycin oral administration for treating Clostridioides difficile infections. Combination therapy with furazolidone, however, is likely to prevent emergence and outgrowth of the lpp-mutated Gram-negative coliforms, avoiding exacerbation of the patient's condition during the treatment.

Robust and Interpretable General Movement Assessment Using Fidgety Movement Detection.

Fidgety movements occur in infants between the age of 9 to 20 weeks post-term, and their absence are a strong indicator that an infant has cerebral palsy. Prechtl's General Movement Assessment method evaluates whether an infant has fidgety movements, but requires a trained expert to conduct it. Timely evaluation facilitates early interventions, and thus computer-based methods have been developed to aid domain experts. However, current solutions rely on complex models or high-dimensional representations of the data, which hinder their interpretability and generalization ability. To address that we propose [Formula: see text], a method that detects fidgety movements and uses them towards an assessment of the quality of an infant's general movements. [Formula: see text] is true to the domain expert process, more accurate, and highly interpretable due to its fine-grained scoring system. The main idea behind [Formula: see text] is to specify signal properties of fidgety movements that are measurable and quantifiable. In particular, we measure the movement direction variability of joints of interest, for movements of small amplitude in short video segments. [Formula: see text] also comprises a strategy to reduce those measurements to a single score that quantifies the quality of an infant's general movements; the strategy is a direct translation of the qualitative procedure domain experts use to assess infants. This brings [Formula: see text] closer to the process a domain expert applies to decide whether an infant produced enough fidgety movements. We evaluated [Formula: see text] on the largest clinical dataset reported, where it showed to be interpretable and more accurate than many methods published to date.

Sequential Organ Failure Assessment (SOFA) Score for predicting mortality in patients with sepsis in Vietnamese intensive care units: a multicentre, cross-sectional study.

OBJECTIVES: To compare the accuracy of the Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation II (APACHE II) Scores in predicting mortality among intensive care unit (ICU) patients with sepsis in a low-income and middle-income country. DESIGN: A multicentre, cross-sectional study. SETTING: A total of 15 adult ICUs throughout Vietnam. PARTICIPANTS: We included all patients aged ≥18 years who were admitted to ICUs for sepsis and who were still in ICUs from 00:00 to 23:59 of the specified study days (ie, 9 January, 3 April, 3 July and 9 October of the year 2019). PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome was hospital all-cause mortality (hospital mortality). We also defined the secondary outcome as all-cause deaths in the ICU (ICU mortality). RESULTS: Of 252 patients, 40.1% died in hospitals, and 33.3% died in ICUs. SOFA Score (areas under the receiver operating characteristic curve (AUROC): 0.688 (95% CI 0.618 to 0.758); cut-off value≥7.5; PAUROC<0.001) and APACHE II Score (AUROC: 0.689 (95% CI 0.622 to 0.756); cut-off value ≥20.5; PAUROC<0.001) both had a poor discriminatory ability for predicting hospital mortality. However, the discriminatory ability for predicting ICU mortality of SOFA (AUROC: 0.713 (95% CI 0.643 to 0.783); cut-off value≥9.5; PAUROC<0.001) was fair and was better than that of APACHE II Score (AUROC: 0.672 (95% CI 0.603 to 0.742); cut-off value≥18.5; PAUROC<0.001). A SOFA Score≥8 (adjusted OR (AOR): 2.717; 95% CI 1.371 to 5.382) and an APACHE II Score≥21 (AOR: 2.668; 95% CI 1.338 to 5.321) were independently associated with an increased risk of hospital mortality. Additionally, a SOFA Score≥10 (AOR: 2.194; 95% CI 1.017 to 4.735) was an independent predictor of ICU mortality, in contrast to an APACHE II Score≥19, for which this role did not. CONCLUSIONS: In this study, SOFA and APACHE II Scores were worthwhile in predicting mortality among ICU patients with sepsis. However, due to better discrimination for predicting ICU mortality, the SOFA Score was preferable to the APACHE II Score in predicting mortality.Clinical trials registry - India: CTRI/2019/01/016898.

The three ghosts of medical AI: Can the black-box present deliver?

Our title alludes to the three Christmas ghosts encountered by Ebenezer Scrooge in A Christmas Carol, who guide Ebenezer through the past, present, and future of Christmas holiday events. Similarly, our article takes readers through a journey of the past, present, and future of medical AI. In doing so, we focus on the crux of modern machine learning: the reliance on powerful but intrinsically opaque models. When applied to the healthcare domain, these models fail to meet the needs for transparency that their clinician and patient end-users require. We review the implications of this failure, and argue that opaque models (1) lack quality assurance, (2) fail to elicit trust, and (3) restrict physician-patient dialogue. We then discuss how upholding transparency in all aspects of model design and model validation can help ensure the reliability and success of medical AI.

Gastric rupture following multiple blunt trauma.

Gastric injury due to trauma is a rare complication that occurs in approximately 0.04%-1.2% of all instances of abdominal trauma. When imaging trauma cases, certain areas can be obscured by several inevitable reasons. Despite its rarity, the high mortality rate of a gastric injury requires an early and accurate diagnosis. We present the case of an 18-year-old male who suffered a gastric rupture of the greater curvature following a road traffic collision before providing a brief review of the literature.

Predictive validity of the quick Sequential Organ Failure Assessment (qSOFA) score for the mortality in patients with sepsis in Vietnamese intensive care units.

BACKGROUND: The simple scoring systems for predicting the outcome of sepsis in intensive care units (ICUs) are few, especially for limited-resource settings. Therefore, this study aimed to evaluate the accuracy of the quick Sequential (Sepsis-Related) Organ Failure Assessment (qSOFA) score in predicting the mortality of ICU patients with sepsis in Vietnam. METHODS: We did a multicenter cross-sectional study of patients with sepsis (≥18 years old) presenting to 15 adult ICUs throughout Vietnam on the specified days (i.e., 9th January, 3rd April, 3rd July, and 9th October) representing the different seasons of 2019. The primary and secondary outcomes were the hospital and ICU all-cause mortalities, respectively. The area under the receiver operating characteristic curve (AUROC) was calculated to determine the discriminatory ability of the qSOFA score for deaths in the hospital and ICU. The cut-off value of the qSOFA scores was determined by the receiver operating characteristic curve analysis. Upon ICU admission, factors associated with the hospital and ICU mortalities were assessed in univariable and multivariable logistic models. RESULTS: Of 252 patients, 40.1% died in the hospital, and 33.3% died in the ICU. The qSOFA score had a poor discriminatory ability for both the hospital (AUROC: 0.610 [95% CI: 0.538 to 0.681]; cut-off value: ≥2.5; sensitivity: 34.7%; specificity: 84.1%; PAUROC = 0.003) and ICU (AUROC: 0.619 [95% CI: 0.544 to 0.694]; cutoff value: ≥2.5; sensitivity: 36.9%; specificity: 83.3%; PAUROC = 0.002) mortalities. However, multivariable logistic regression analyses show that the qSOFA score of 3 was independently associated with the increased risk of deaths in both the hospital (adjusted odds ratio, AOR: 3.358; 95% confidence interval, CI: 1.756 to 6.422) and the ICU (AOR: 3.060; 95% CI: 1.651 to 5.671). CONCLUSION: In our study, despite having a poor discriminatory value, the qSOFA score seems worthwhile in predicting mortality in ICU patients with sepsis in limited-resource settings. CLINICAL TRIAL REGISTRATION: Clinical trials registry-India: CTRI/2019/01/016898.

Trust and medical AI: the challenges we face and the expertise needed to overcome them.

Artificial intelligence (AI) is increasingly of tremendous interest in the medical field. How-ever, failures of medical AI could have serious consequences for both clinical outcomes and the patient experience. These consequences could erode public trust in AI, which could in turn undermine trust in our healthcare institutions. This article makes 2 contributions. First, it describes the major conceptual, technical, and humanistic challenges in medical AI. Second, it proposes a solution that hinges on the education and accreditation of new expert groups who specialize in the development, verification, and operation of medical AI technologies. These groups will be required to maintain trust in our healthcare institutions.

In vitro synergy of 5-nitrofurans, vancomycin and sodium deoxycholate against Gram-negative pathogens.

Introduction. There is an urgent need for effective therapies against bacterial infections, especially those caused by antibiotic-resistant Gram-negative pathogens.Hypothesis. Synergistic combinations of existing antimicrobials show promise due to their enhanced efficacies and reduced dosages which can mitigate adverse effects, and therefore can be used as potential antibacterial therapy.Aim. In this study, we sought to characterize the in vitro interaction of 5-nitrofurans, vancomycin and sodium deoxycholate (NVD) against pathogenic bacteria.Methodology. The synergy of the NVD combination was investigated in terms of growth inhibition and bacterial killing using checkerboard and time-kill assays, respectively.Results. Using a three-dimensional checkerboard assay, we showed that 5-nitrofurans, sodium deoxycholate and vancomycin interact synergistically in the growth inhibition of 15 out of 20 Gram-negative strains tested, including clinically significant pathogens such as carbapenemase-producing Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii, and interact indifferently against the Gram-positive strains tested. The time-kill assay further confirmed that the triple combination was bactericidal in a synergistic manner.Conclusion. This study demonstrates the synergistic effect of 5-nitrofurans, sodium deoxycholate and vancomycin against Gram-negative pathogens and highlights the potential of the combination as a treatment for Gram-negative and Gram-positive infections.