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Additional file 1: Table S1. List of primary antibodies used for immunofluorescence studies and Western blotting experiments. Figure S1. Thrombin [5U] and human albumin [10 nM] was used as a positive and negative control, respectively, for Dextran-FITC permeability of the BBB. The fluorescence intensity of Dextran-FITC was measured in the lower chamber. The data represent the mean ± SEM of four independent experiments (N=4, two inserts per condition each time). * and ^ indicate statistical significance at p ≤ 0.05 compared to CT and S1, respectively by one-way ANOVA and Tukey post hoc test. Figure S2. Western blot bands (Panel A) and VE-cadherin and β-catenin expression fold change (Panel B). The data represent the mean ± SEM of four independent experiments (N=4). * and ^ indicate statistical significance at p ≤ 0.05 compared to CT and S1, respectively by one-way ANOVA and Tukey post hoc test. Figure S3. Log2FC of gene expression from Fig. 7A. Statistical significance at p ≤ 0.05 was assessed by one-way ANOVA and Tukey post hoc test. Figure S4. Raw images of Western blot.

Sylwia Joanna Chmielewska,1 Karol Skłodowski,1 Ewelina Piktel,1 Łukasz Suprewicz,1 Krzysztof Fiedoruk,1 Tamara Daniluk,1 Przemysław Wolak,2 Paul B Savage,3 Robert Bucki1 1Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of Białystok, Białystok, Poland; 2The Faculty of Medicine and Health Sciences of the Jan Kochanowski University in Kielce, Kielce, Poland; 3Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USACorrespondence: Robert BuckiDepartment of Medical Microbiology and Nanobiomedical Engineering, Medical University of Białystok, Mickiewicza 2C, Białystok 15-222, PolandTel +48 85 748 5493Fax +48 85 748 5416Email buckirobert@gmail.comBackground and Purpose: Treatment of infections caused by NDM-1 carbapenemase-producing Enterobacteriaceae (CPE) represents one of the major challenges of modern medicine. In order to address this issue, we tested ceragenins (CSAs – cationic steroid antimicrobials) as promising agents to eradicate various NDM-1-producing Gram-negative enteric rods.Materials and Methods: Susceptibility to CSA-13, CSA-44, and CSA-131 of four reference NDM-1 carbapenemase-producing strains, ie, Escherichia coli BAA-2471, Enterobacter cloacae BAA-2468, Klebsiella pneumoniae subsp. pneumoniae BAA-2472, and K. pneumoniae BAA-2473 was assessed by MIC/MBC testing of planktonic cells as well as biofilm formation/disruption assays. To define the mechanism of CSAs bactericidal activity, their ability to induce generation of reactive oxygen species (ROS), permeabilization of the inner and outer membranes, and their mechanical and adhesive properties upon CSA addition were examined. Additionally, hemolytic assays were performed to assess CSAs hemocompatibility.Results: All tested CSAs exert substantial bactericidal activity against NDM-1-producing bacteria. Moreover, CSAs significantly prevent biofilm formation as well as reduce the mass of developed biofilms. The mechanism of CSA action comprises both increased permeability of the outer and inner membrane, which is associated with an extensive ROS generation. Additionally, atomic force microscopy (AFM) analysis has shown morphological alterations in bacterial cells and the reduction of stiffness and adhesion properties. Importantly, CSAs are characterized by low hemolytic activity at concentrations that are bactericidal.Conclusion: Development of ceragenins should be viewed as one of the valid strategies to provide new treatment options against infections associated with CPE. The studies presented herein demonstrate that NDM-1-positive bacteria are more susceptible to ceragenins than to conventional antibiotics. In effect, CSA-13, CSA-44, and CSA-131 may be favorable for prevention and decrease of global burden of CPE.Keywords: antibiotic resistance, NDM-1, CSA-13, CSA-44, CSA-131, ceragenins, CPE

Additional file 1: Table S1. C. jejuni ST50, ST51 and ST257 genomes (n = 116) collected in the PATRIC genome database. Table S2. C. jejuni virulence genes (n = 143) under the study. Table S3. The twelve most common STs among C. jejuni genomes (n = 116) deposited in the PATRIC genome database. Table S4. The occurrence of virulence genes (n = 143) in C. jejuni ST50, ST51 and ST257 strains under the study. Table S5. The occurence of 25 genes recognized by Buchanan et al. [17] as markers of human pathogenic C. jejuni strains in genomes of C. jejuni ST50, ST51 and ST257. Figure S1. The distribution C. jejuni pan-genome genes and the number of isolates possessing them (visualized using roary_plots.py script). Figure S2. The frequency of C. jejuni genes versus the number of genomes (visualized using roary_plots.py script). Figure S3. The phylogenetic tree compared to a matrix with the presence and absence of C. jejuni core and accessory genes (visualized using roary_plots.py script). Figure S4. Similarity of C. jejuni 81-176 plasmid pTet (A) and C. jejuni CFSAN054107 plasmid pGMI16-002 (B) with C. jejuni strains under the study carrying tetO on plasmid contigs. Figure S5. UPGMA cluster tree illustrating identity of amino acid sequences of TetO variants detected in C. jejuni strains. The evolutionary distances were computed using the Poisson correction method, and are in the units of the number of amino acid substitutions per site. The tree was built in MEGA7 software. Figure S6. Chromosomal locations of the tetO gene in C. jejuni genomes under the study. Figure S7. Similarity of C. jejuni CFSAN054107 plasmid pGMI16-002 with C. jejuni genomes under the study carrying aminoglycoside resistance genes.