Journal: Nature Communications

Article Title: Human neutrophil α-defensin HNP1 interacts with bacterial OmpA to promote Acinetobacter baumannii biofilm formation

doi: 10.1038/s41467-025-60935-7

Figure Lengend Snippet: A HNP1 promoted biofilm formation by A. baumannii 19606 in a dose-dependent manner. Results are normalized to the control group (0 μM) and represent the mean ± SD of at least three independent experiments ( n = 3 biological replicates per experiment). B HNP1 did not inhibit bacterial growth in LB broth (LB, orange), while exhibited significant bactericidal activity in 10 mM phosphate buffer (PB, gray). Bactericidal activity was evaluated using VCC assays. Results are mean ± SD, representative of two independent experiments ( n = 3 biological replicates per experiment). C Visualization of biofilm formation by GFP-expressing A. baumannii on silicone pieces using optical microscopy after crystal violet staining (CV, scale bar = 25 μm), confocal microscopy (GFP, scale bar = 20 μm), and scanning electron microscopy (SEM, scale bar = 10 μm). Six-μm-thick z stacks from confocal microscopies were reconstructed in three-dimension using Imaris Viewer software (3D). Images are representative of at least two independent experiments. D Schematic illustration of biofilm observation using the BioFlux 1000z system (redrawn from a schematic from the BioFlux manufacturer). Diluted inoculum was applied to the 48-well plate channel with 0, 4 or 8 μM HNP1, followed by microscopic photographing continuously under a flow condition. E Real-time monitoring of biofilm formation in the Bioflux 1000z system. F Quantification of biofilms in the channels as mean gray values using ImageJ. Results represent mean with 95% confidence intervals from five parallels. G Heatmap showing the enhancement of biofilm formation by HNP1 across various A. baumannii clinical isolates, quantified by CV staining. Results are representative of at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA. H Comparative effects of different antimicrobial peptides (4 μM) on biofilm formation, analyzed using CV staining. Results are violin plot of three independent experiments ( n = 3 biological replicates per experiment). Statistical significances ( A , B , G and H ) were evaluated by one-way ANOVA with Tukey correction for multiple comparison. See also Supplementary Figs. , , Supplementary Table and Supplementary Movie .

Article Snippet: The Acinetobacter baumannii standard strain ATCC 19606, its OmpA knockout mutant (ΔOmpA) , and the complementary strain (ΔOmpA:C) , as well as the GFP-expressing 19606 strain , were generously provided by Prof. Luis A. Actis from Miami University.

Techniques: Control, Activity Assay, Expressing, Microscopy, Staining, Confocal Microscopy, Electron Microscopy, Software, Comparison

Journal: Nature Communications

Article Title: Human neutrophil α-defensin HNP1 interacts with bacterial OmpA to promote Acinetobacter baumannii biofilm formation

doi: 10.1038/s41467-025-60935-7

Figure Lengend Snippet: A Adhesion of three 19606 strains on A549 cells evaluated by the bacterial adhesion assay. The cells were washed with DMEM, incubated with 19606 in the presence or absence of HNP1 for 2 h, lysed with water, diluted with PBS, and then plated on LB agar for colony counting after overnight culture. B Effect of HNP1 on biofilm formation of three 19606 strains. Upper panel: Biofilm formation in 96-well plates quantified by CV staining. Lower panel: Biofilm formation on the air-liquid interface of polystyrene tubes stained with CV. HNP1 did not enhance bacterial adhesion to A549 cells ( A ) or biofilm formation ( B ) in the ompA knockout mutant (ΔOmpA), but these effects were restored with ompA complementation (ΔOmpA:C). Results of A and B are mean ± SD representative of at least two experiments ( n = 6 biological replicates per experiment). C Visualization using fluorescent microscopy of biofilm formation on silicone surfaces by three A. baumannii 19606 strains with/without HNP1, stained by PI (Scale bar = 50 μm), representative of two independent experiments. D Visualization using SEM of biofilm formation on silicone surfaces by A. baumannii 19606 WT and ΔOmpA strains with/without HNP1 (Scale bar = 50 μm), representative of two biological replicates. E Competitive inhibition of exogenously added recombinant OmpA on HNP1-promoted biofilm formation. Results are pooled mean ± SD of three independent experiments ( n = 3 biological replicates per experiment). Statistical significances ( A , B and E ) were evaluated by two-way ANOVA with Tukey correction for multiple comparison. See also Supplementary Figs. and .

Article Snippet: The Acinetobacter baumannii standard strain ATCC 19606, its OmpA knockout mutant (ΔOmpA) , and the complementary strain (ΔOmpA:C) , as well as the GFP-expressing 19606 strain , were generously provided by Prof. Luis A. Actis from Miami University.

Techniques: Cell Adhesion Assay, Incubation, Staining, Knock-Out, Mutagenesis, Microscopy, Inhibition, Recombinant, Comparison

Journal: Nature Communications

Article Title: Human neutrophil α-defensin HNP1 interacts with bacterial OmpA to promote Acinetobacter baumannii biofilm formation

doi: 10.1038/s41467-025-60935-7

Figure Lengend Snippet: A The structure of A. baumannii OmpA constructed using AlphaFold 3. The transmembrane domain is in green, the peptidoglycan-binding domain in the periplasm is in yellow, and the two extracellular loops (L1 and L2) are in red. B Binding affinity between the full-length wild-type OmpA (OmpA WT) and HNP1, as well as the OmpA ΔL1L2 mutant (OmpA-ΔL1L2) and HNP1, assessed by fluorescent polarization. C Binding affinity between the transmembrane domain of wild-type OmpA (TM) and HNP1, as well as the OmpA ΔL1L2 mutant (TM-ΔL1L2) and HNP1, assessed by fluorescent polarization. Results of B and C are mean ± SD representative of at least two experiments ( n = 2 biological replicates per experiment). D Conformations at different time points of HNP1 and OmpA in molecular dynamics simulation. OmpA is shown in green, interacting HNP1 molecules (M1 and M2) in red, and diffusing HNP1 molecules (M3 and M4) in gray. E The decomposition of individual residue contributions of OmpA in the total binding free energy during 100–1000 ns of MD simulation analyzed using MM-GBSA. F Effect of HNP1 Ala-substitution mutants on biofilm formation characterized by CV staining. Results are mean ± SD of two independent experiments (three parallels per experiment). G Position of critical residues on an HNP1 dimer (PDB: 3GNY). H Restorage of hydrophobicity at position 26 of HNP1 restored its biofilm-enhancing effect. Results are violin plot of four independent experiments (three parallels per experiment). Statistical significances ( F and H ) were evaluated by one-way ANOVA with Tukey correction for multiple comparison. See also Supplementary Figs. – and Supplementary Movie .

Article Snippet: The Acinetobacter baumannii standard strain ATCC 19606, its OmpA knockout mutant (ΔOmpA) , and the complementary strain (ΔOmpA:C) , as well as the GFP-expressing 19606 strain , were generously provided by Prof. Luis A. Actis from Miami University.

Techniques: Construct, Binding Assay, Mutagenesis, Residue, Staining, Comparison

Journal: Nature Communications

Article Title: Human neutrophil α-defensin HNP1 interacts with bacterial OmpA to promote Acinetobacter baumannii biofilm formation

doi: 10.1038/s41467-025-60935-7

Figure Lengend Snippet: A PCA plot of RNA-seq data from A. baumannii WT and ΔOmpA strains treated with/without 8 μM HNP1 ( n = 3 for each group). B Hierarchically clustered heatmap representing sample-to-sample distances calculated using Poisson distances, illustrating the overall similarities between samples. C Venn diagrams showing the overlap of DEGs between the three comparisons: WT + HNP1 vs. WT, ΔOmpA vs. WT, and ΔOmpA + HNP1 vs. ΔOmpA. The upper panel shows overlaps of upregulated genes, while the lower panel shows overlaps of downregulated genes. D Volcano plots depicting the DEGs from the DESeq2 analysis for the three comparisons. Genes significantly upregulated are shown in red, while significantly downregulated genes are shown in blue. Non-significant genes are indicated in gray. E COG category distribution of DEGs. The upregulated and downregulated genes in each comparison are categorized by their COG functional groups, with blue representing downregulated genes and orange representing upregulated genes. F Heatmap showing the expression of nine-groups of biofilm-related genes across the four experimental groups. The upper panel displays the mean log2 fold change relative to WT for each gene, and the lower panel shows the count per million (CPM) expression values for the same genes. See also Supplementary Figs. and , .

Article Snippet: The Acinetobacter baumannii standard strain ATCC 19606, its OmpA knockout mutant (ΔOmpA) , and the complementary strain (ΔOmpA:C) , as well as the GFP-expressing 19606 strain , were generously provided by Prof. Luis A. Actis from Miami University.

Techniques: RNA Sequencing, Comparison, Functional Assay, Expressing

Journal: Frontiers in Microbiology

Article Title: A multiplex RPA coupled with CRISPR-Cas12a system for rapid and cost-effective identification of carbapenem-resistant Acinetobacter baumannii

doi: 10.3389/fmicb.2024.1359976

Figure Lengend Snippet: Source of bacterial strains used in this study and carbapenem resistance.

Article Snippet: Standard strains of A. baumannii (ATCC 19606), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853), and Stenotrophomonas maltophilia (ATCC 19861) were provided by our laboratory strain bank.

Techniques: Isolation

Journal: Frontiers in Microbiology

Article Title: A multiplex RPA coupled with CRISPR-Cas12a system for rapid and cost-effective identification of carbapenem-resistant Acinetobacter baumannii

doi: 10.3389/fmicb.2024.1359976

Figure Lengend Snippet: Detection of clinical strains of A. baumannii . NC, negative target control. S1-S30, A. baumannii obtain from clinical. M, Marker. (A) Agarose gel image showing the clinical strains tested with the PCR assay. (B,C) show the visualised signals and the heatmap of endpoint fluorescence values of 30 clinical strains detected using multiplex RPA-CRISPR-Cas12a.

Article Snippet: Standard strains of A. baumannii (ATCC 19606), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853), and Stenotrophomonas maltophilia (ATCC 19861) were provided by our laboratory strain bank.

Techniques: Control, Marker, Agarose Gel Electrophoresis, Fluorescence, Multiplex Assay, CRISPR

Journal: Current Research in Microbial Sciences

Article Title: Identification and characterization of amphipathic antimicrobial peptides with broad spectrum activity against multi-drug resistant bacteria.

doi: 10.1016/j.crmicr.2025.100363

Figure Lengend Snippet: Bacterial strains used to test antimicrobial activity.

Article Snippet: Time-killing curves represent CFU counts in cultures at 0, 3, 6 and 24 h of the strains A. baumannii ATCC 19,606 (A), K. pneumoniae CG258 (B) and E. coli ESBL 1057.1 (C) in the presence of MIC (square) and 2× MIC (triangle) of peptides BP607 (white), BP145 (grey) and BP76 (black).

Techniques: Activity Assay, Control

Journal: Current Research in Microbial Sciences

Article Title: Identification and characterization of amphipathic antimicrobial peptides with broad spectrum activity against multi-drug resistant bacteria.

doi: 10.1016/j.crmicr.2025.100363

Figure Lengend Snippet: Minimum inhibitory concentration (MIC) and Minimum Bactericidal Concentration (MBC) of peptides BP76, BP145 and BP607 against the different bacterial strains.

Article Snippet: Time-killing curves represent CFU counts in cultures at 0, 3, 6 and 24 h of the strains A. baumannii ATCC 19,606 (A), K. pneumoniae CG258 (B) and E. coli ESBL 1057.1 (C) in the presence of MIC (square) and 2× MIC (triangle) of peptides BP607 (white), BP145 (grey) and BP76 (black).

Techniques: Concentration Assay

Journal: Current Research in Microbial Sciences

Article Title: Identification and characterization of amphipathic antimicrobial peptides with broad spectrum activity against multi-drug resistant bacteria.

doi: 10.1016/j.crmicr.2025.100363

Figure Lengend Snippet: Time-killing assays for BP76, BP145, and BP607. Time-killing curves represent CFU counts in cultures at 0, 3, 6 and 24 h of the strains A. baumannii ATCC 19,606 (A), K. pneumoniae CG258 (B) and E. coli ESBL 1057.1 (C) in the presence of MIC (square) and 2× MIC (triangle) of peptides BP607 (white), BP145 (grey) and BP76 (black). Time-killing curves in MHBII (circles) were carried out as a control.

Article Snippet: Time-killing curves represent CFU counts in cultures at 0, 3, 6 and 24 h of the strains A. baumannii ATCC 19,606 (A), K. pneumoniae CG258 (B) and E. coli ESBL 1057.1 (C) in the presence of MIC (square) and 2× MIC (triangle) of peptides BP607 (white), BP145 (grey) and BP76 (black).

Techniques: Control

Journal: Microbial Genomics

Article Title: Genome diversity of domesticated Acinetobacter baumannii ATCC 19606 T strains

doi: 10.1099/mgen.0.000749

Figure Lengend Snippet: Chromosome map of A. baumannii ATCC 19606(A). Circular map created by the CGView server. From the outermost to innermost, the tracks show the genes on positive (dark blue) and negative (light blue) strands, ORFs on positive and negative strands (with colours indicating COG classifications; ), prophages (red) with dotted lines indicating the excision site of the missing prophage, GIs (orange), GC content (green) and GC skew (purple and light green for positive and negative, respectively). Position 1 in ATCC 19606(A) corresponds to position 3772737 in ATCC 19696(H) and position 1094161 in ATCC 19606(M). Both genomes are in reverse orientation relative to ATCC 19606(A).

Article Snippet: The ATCC 19606(A) chromosome and plasmid sequences are now publicly available under GenBank Accession numbers of CP058289, CP058290 and CP058291, respectively.

Techniques:

Journal: Microbial Genomics

Article Title: Genome diversity of domesticated Acinetobacter baumannii ATCC 19606 T strains

doi: 10.1099/mgen.0.000749

Figure Lengend Snippet: A. baumannii Φ19606 phage. (a) Circular map of the Φ19606 genome drawn with DNAPlotter. The genome map illustrates putative ORFs along with the direction of transcription indicated with arrows. Functional proteins predicted by PHASTER are depicted in different colours. (b) Integration site of Φ19606 (black) into the ATCC 19606(M, D, H) chromosomes (top). The double slash denotes a phage region that is not shown. Positions refer to the ATCC 19606(M) genome sequence. Structure of ATCC 19606(A, S, T) after phage loss (bottom). Positions refer to ATCC 19606(A) genome sequence. Sequences flanking the insertion site are boxed, with predicted phage nucleotides italicized. Primer positions are indicated with black arrows. N 60 stands for the 60-nucleotide sequence generated by phage insertion/excision. (c) Agarose gel electrophoresis of the PCR products obtained by using different primer pairs indicated in (b). (d) Presence (+) or absence (-) of amplicons detected in the different A. baumannii ATCC 19606 T strains.

Article Snippet: The ATCC 19606(A) chromosome and plasmid sequences are now publicly available under GenBank Accession numbers of CP058289, CP058290 and CP058291, respectively.

Techniques: Functional Assay, Sequencing, Generated, Agarose Gel Electrophoresis

Journal: Microbial Genomics

Article Title: Genome diversity of domesticated Acinetobacter baumannii ATCC 19606 T strains

doi: 10.1099/mgen.0.000749

Figure Lengend Snippet: Plasmids p1ATCC19606 and pMAC harboured by A. baumannii ATCC 19606 T strains. (a) Agarose gel electrophoresis of clear lysates of A. baumannii ATCC 19606(A) (lane 1), ATCC 19606(D) (lane 2), ATCC 19606(S) (lane 3) and ATCC 19606(T) (lane 4). M, Lambda DNA/HindIII marker (ThermoFisher). White arrows indicate the closed circular forms of pMAC (upper band) and p1ATCC19606 (lower band). (b) p1ATCC19606 and pMAC were copurified from A. baumannii strains ATCC 19606(A) (lanes 1 and 5), ATCC 19606(D) (lanes 2 and 6), ATCC 19606(S) (lanes 3 and 7) and ATCC 19606(T) (lanes 4 and 8), and digested with XhoI (lanes 1–4) and BclI (lanes 5–8). M, BenchTop 1 kb DNA Ladder (Promega). (c) Physical and functional maps of the p1ATCC19606 and pMAC plasmids. Restriction sites for the enzymes used to generate the electropherogram in (b) are shown. Unique cutter restriction enzymes are indicated in bold. Nomenclature of p1ATCC19606: rep , putative replicase; dbp , gene encoding a predicted DNA-binding protein; cspE -like, putative cold-shock protein gene; sel1 -like, putative gene coding for a Sel1-repeat family protein; yedL -like, gene coding for the putative YedL N-acetyltransferase; oriC, predicted origin of replication. Nomenclature of pMAC: repM , replication protein M; dbp , gene encoding a predicted DNA-binding protein; ohr , gene encoding an organic hydroperoxide resistance protein, mobA , plasmid mobilization protein; oriC, origin of replication. ORFs shown in black are predicted to encode for hypothetical proteins. All genes are reported in scale over the total length of each plasmid. Images were obtained by the use of the SnapGene software (GSL Biotech).

Article Snippet: The ATCC 19606(A) chromosome and plasmid sequences are now publicly available under GenBank Accession numbers of CP058289, CP058290 and CP058291, respectively.

Techniques: Agarose Gel Electrophoresis, Lambda DNA Preparation, Marker, Functional Assay, Binding Assay, Plasmid Preparation, Software