Journal: BMC Complementary and Alternative Medicine

Article Title: Steroidal alkaloids and conessine from the medicinal plant Holarrhena antidysenterica restore antibiotic efficacy in a Galleria mellonella model of multidrug-resistant Pseudomonas aeruginosa infection

doi: 10.1186/s12906-018-2348-9

Figure Lengend Snippet: MICs of five antibiotics alone and in the presence of Holarrhena antidysenterica steroidal alkaloids or conessine against P. aeruginosa PAM1020, 1032, 1033, 1034 and 1626

Article Snippet: P. aeruginosa strains PAM1020 (PAO1 prototroph, wild-type); PAM1032 ( nalB ), MexAB-OprM overexpressed; PAM1033 ( nfxB ), MexCD-OprJ overexpressed; PAM1034 ( nfxC ), MexEF-OprN overexpressed; and PAM1626 ( ∆mex ), MexAB-OprM, MexCD-OprJ and MexEF-OprN deletion, were a generous gift from Dr. Olga Lomovskaya, Rempex Pharmaceuticals, USA [ ].

Techniques:

Journal: BMC Complementary and Alternative Medicine

Article Title: Steroidal alkaloids and conessine from the medicinal plant Holarrhena antidysenterica restore antibiotic efficacy in a Galleria mellonella model of multidrug-resistant Pseudomonas aeruginosa infection

doi: 10.1186/s12906-018-2348-9

Figure Lengend Snippet: Effect of conessine and steroidal alkaloids in combination with levofloxacin on the viability of P. aeruginosa PAM1020, PAM1032 and PAM1034

Article Snippet: P. aeruginosa strains PAM1020 (PAO1 prototroph, wild-type); PAM1032 ( nalB ), MexAB-OprM overexpressed; PAM1033 ( nfxB ), MexCD-OprJ overexpressed; PAM1034 ( nfxC ), MexEF-OprN overexpressed; and PAM1626 ( ∆mex ), MexAB-OprM, MexCD-OprJ and MexEF-OprN deletion, were a generous gift from Dr. Olga Lomovskaya, Rempex Pharmaceuticals, USA [ ].

Techniques: Control, Concentration Assay

Journal: BMC Complementary and Alternative Medicine

Article Title: Steroidal alkaloids and conessine from the medicinal plant Holarrhena antidysenterica restore antibiotic efficacy in a Galleria mellonella model of multidrug-resistant Pseudomonas aeruginosa infection

doi: 10.1186/s12906-018-2348-9

Figure Lengend Snippet: Effect of treatment with combinations of conessine and levofloxacin on survival of G. mellonella larvae infected with P. aeruginosa PAM1020 ( a ), PAM1032 ( b ) and PAM1034 ( c ), or larval burden of the same strains PAM1020 ( d ), PAM1032 ( e ) and PAM1034 ( f ). All larvae were inoculated with 2.5 × 10 3 cfu/mL P. aeruginosa and treated with each agent individually or in combination with three doses at 2, 4 and 6 h post-infection (indicated by the arrows). Treatments consisted of PBS, conessine (50 mg/kg), levofloxacin (1 or 5 mg/kg, indicated on graph), and a combination of conessine with levofloxacin. Larvae were incubated at 37 °C for 96 h and survival recorded every 24 h. The burden of P. aeruginosa was determined from five individual larvae every 24 h. For clarity, data for treatment with PBS alone is not shown because the data obtained was similar to that obtained for conessine treatment alone. * a ), b ) and c ); combination treatment group with significantly enhanced survival compared with any of the constituent monotherapies ( P < 0.05, log-rank test with Holm’s correction for multiple comparisons). n = 45 (pooled from triplicate experiments). Error bars indicate ±SEM. LVX, levofloxacin; CON, conessine. * d ), e ) and f ); significant difference in larval burden between groups treated with the combination of conessine and levofloxacin compared with the constituent monotherapies; n = 5 ( P < 0.05, the Mann–Whitney U -test compared the combination therapy with each monotherapy individually). The black bar represents the median value of larval burden per group

Article Snippet: P. aeruginosa strains PAM1020 (PAO1 prototroph, wild-type); PAM1032 ( nalB ), MexAB-OprM overexpressed; PAM1033 ( nfxB ), MexCD-OprJ overexpressed; PAM1034 ( nfxC ), MexEF-OprN overexpressed; and PAM1626 ( ∆mex ), MexAB-OprM, MexCD-OprJ and MexEF-OprN deletion, were a generous gift from Dr. Olga Lomovskaya, Rempex Pharmaceuticals, USA [ ].

Techniques: Infection, Incubation, MANN-WHITNEY

Journal: Microbiology Spectrum

Article Title: Evaluating Bacterial Pathogenesis Using a Model of Human Airway Organoids Infected with Pseudomonas aeruginosa Biofilms

doi: 10.1128/spectrum.02408-22

Figure Lengend Snippet: Modeling P. aeruginosa infection on HAOs. Histology of healthy human airway epithelium (A) and HAOs at the air-liquid interface (ALI) stained with H&E (B). (C) Immunofluorescent (IF) detection of acetyl-α-tubulin, MUC5AC, p63α, CFTR, and CLDN4 in the HAOs, with nuclei stained using DAPI. Scale bars = 10, 20, or 50 μm. (D) P. aeruginosa expansion imaged using fluorescence microscopy at 3, 6, 12, and 24 hpi. Red, P. aeruginosa strains labeled with a lac ::mCherry plasmidic fusion. Scale bar = 50 μm. (E) Bacterial growth curve was measured as the number of CFU recovered from HAOs. Data are shown as mean ± SEM from three independent experiments done in triplicate. P values were calculated by one-way analysis of variance (ANOVA) with Tukey’s posttest for multiple comparisons.

Article Snippet: Next, 100-μL aliquots of the eluted solution were used as the culture medium for P. aeruginosa strains to grow biofilm for 24 h in 96-well plates (Corning).

Techniques: Infection, Staining, Fluorescence, Microscopy, Labeling

Journal: Microbiology Spectrum

Article Title: Evaluating Bacterial Pathogenesis Using a Model of Human Airway Organoids Infected with Pseudomonas aeruginosa Biofilms

doi: 10.1128/spectrum.02408-22

Figure Lengend Snippet: Global transcriptomic analysis of P. aeruginosa on the HAOs at 24 hpi. (A) PCA plot for differentially expressed genes in all of the P. aeruginosa samples. (B) Number of genes differentially expressed in the different paired groups. Red columns, numbers of upregulated genes; blue columns, numbers of downregulated genes. (C) Heat map of differentially expressed genes, normalized for sequencing depth across all samples. The top 30 enriched KEGG pathways are listed on the left. (D) Volcano plot of differentially expressed genes in PAO1 WT grown on HAOs, relative to PAO1 in LB medium.

Article Snippet: Next, 100-μL aliquots of the eluted solution were used as the culture medium for P. aeruginosa strains to grow biofilm for 24 h in 96-well plates (Corning).

Techniques: Sequencing

Journal: Microbiology Spectrum

Article Title: Evaluating Bacterial Pathogenesis Using a Model of Human Airway Organoids Infected with Pseudomonas aeruginosa Biofilms

doi: 10.1128/spectrum.02408-22

Figure Lengend Snippet: Global transcriptomic analysis of HAOs that were exposed to P. aeruginosa at 24 hpi. (A) Number of genes that were differentially expressed in different paired groups. Red columns, numbers of upregulated genes; blue columns, numbers of downregulated genes. (B) PCA plot for differentially expressed genes in all the HAO samples. (C) Heat map of differentially expressed genes, normalized for sequencing depth across all samples. The top 60 enriched KEGG pathways are listed on the left. (D) Differential expression analysis of WT exposed and unexposed HAO cells. Differentially expressed genes (adjusted P < 0.01) are highlighted in blue (log 2 [fold change] ≤ −1, downregulated) and pink (log 2 [fold change] ≥ 1, upregulated); genes relevant to regulation of TJs and the actin cytoskeleton and the NF-κB signaling pathway are specifically labeled. (E) Several inflammation/apoptosis-associated immune responses were upregulated in the PAO1 WT-infected cells, relative to the unexposed cells. Normalized enrichment score (NES) for gene sets significantly enriched (adjusted P value) in each comparison are shown, with positive values denoting upregulation and negative values representing downregulation.

Article Snippet: Next, 100-μL aliquots of the eluted solution were used as the culture medium for P. aeruginosa strains to grow biofilm for 24 h in 96-well plates (Corning).

Techniques: Sequencing, Expressing, Labeling, Infection

Journal: Microbiology Spectrum

Article Title: Evaluating Bacterial Pathogenesis Using a Model of Human Airway Organoids Infected with Pseudomonas aeruginosa Biofilms

doi: 10.1128/spectrum.02408-22

Figure Lengend Snippet: P. aeruginosa biofilm grown on HAOs at 24 hpi. (A) (Left) Volumetric projection of representative images of P. aeruginosa biofilm. (Right) Projections of XZ (blue-green crossline), XY (red-green crossline), and YZ (blue-red crossline) of the corresponding images. Scale bar = 50 μm. The biomass (B) and maximum height (C) of the P. aeruginosa biofilm grown on HAOs at 24 hpi were quantified using the Comstat program. Data are shown as mean ± SEM from three independent experiments. P values were calculated by one-way ANOVA with Tukey’s posttest for multiple comparisons.

Article Snippet: Next, 100-μL aliquots of the eluted solution were used as the culture medium for P. aeruginosa strains to grow biofilm for 24 h in 96-well plates (Corning).

Techniques:

Journal: Microbiology Spectrum

Article Title: Evaluating Bacterial Pathogenesis Using a Model of Human Airway Organoids Infected with Pseudomonas aeruginosa Biofilms

doi: 10.1128/spectrum.02408-22

Figure Lengend Snippet: Cellular architecture of HAOs disrupted by P. aeruginosa infection. (A) TEER significantly decreased over time after exposure of the HAO apical surface to the P. aeruginosa strain PAO1 or its isogenic QS mutants. Values were presented as means ± SEM; P values were calculated by one-way ANOVA with Tukey’s posttest, compared with the PAO1 WT strain. (B and C) H&E-stained histopathology of tangential sections of the HAOs that had been subjected to treatment with PBS or invasion of P. aeruginosa PAO1 for 24 h. HAO sections were stained using immunofluorescence with antibodies of acetyl-α-tubulin and MUC5AC (D), Claudin 4 (E), and Claudin 3 (F). Nuclei were counterstained using DAPI. Sizes of scale bars are indicated in the images.

Article Snippet: Next, 100-μL aliquots of the eluted solution were used as the culture medium for P. aeruginosa strains to grow biofilm for 24 h in 96-well plates (Corning).

Techniques: Infection, Staining, Histopathology, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Elimination of Pseudomonas aeruginosa through Efferocytosis upon Binding to Apoptotic Cells

doi: 10.1371/journal.ppat.1006068

Figure Lengend Snippet: Cell junction types and P . aeruginosa adhesion sites were analyzed by quantitative confocal microscopy. (A) Representative micrograph showing different cell junctions types: Junction of five cells (full arrowhead), junction of four cells (arrow), junction of three cells (dotted square) and junction of two cells (dotted ellipse). The numbers indicate the cells that come together to form a junction of five. (B) The gray bars (left, y-axis) represent the frequency of junction types, based on the analysis of four randomly imaged regions (~300 cells/region) of a polarized MDCK monolayer. The black circles (right, y-axis) represent the number of P . aeruginosa aggregates formed per junction type normalized to the junction type frequency. Data are presented as mean ± SEM. Pearson correlation coefficient r = 0.95, p< 0.05. (C) Projected confocal Z stacks showing examples of P . aeruginosa adhered to multicellular junctions formed by four (left) and five cells (right). PAK-GFP: green and F-actin: red. Scale bars: 10 μm.

Article Snippet: While we were preparing this manuscript, Schwarzer et al. reported that P . aeruginosa strain PA01 migrates to dying cells in scratch-wounded epithelia and that some bacteria remain immobilized on dead cells [ ].

Techniques: Confocal Microscopy

Journal: PLoS Pathogens

Article Title: Elimination of Pseudomonas aeruginosa through Efferocytosis upon Binding to Apoptotic Cells

doi: 10.1371/journal.ppat.1006068

Figure Lengend Snippet: (A) Projected confocal Z stacks and orthogonal sections (lower images) of transwell-grown monolayers infected with PAK. P . aeruginosa adhered to an extruded apoptotic cell (left) and to an apoptotic body (right). PAK-GFP: green, F-actin: red and Annexin V: blue. (B) Glass-grown monolayers infected with PAK. Apoptotic cells to which P . aeruginosa adheres have active Caspase 3. PAK-mCherry: red, Annexin V: green and active Caspase-3: blue. Brightfield depicts the monolayer underneath. (C) Percentage of aggregates formed at Annexin V-positive sites. (D) Percentage of Annexin V-positive spots with at least one adhered bacterium. (E) Percentage of aggregates formed at active Caspase 3-positive sites. Data are presented as mean ± SEM. * p <0.05, Student’s t -test. Scale bars: 10 μm.

Article Snippet: While we were preparing this manuscript, Schwarzer et al. reported that P . aeruginosa strain PA01 migrates to dying cells in scratch-wounded epithelia and that some bacteria remain immobilized on dead cells [ ].

Techniques: Infection

Journal: PLoS Pathogens

Article Title: Elimination of Pseudomonas aeruginosa through Efferocytosis upon Binding to Apoptotic Cells

doi: 10.1371/journal.ppat.1006068

Figure Lengend Snippet: (A) Proportion of internalized apoptotic material (Intracellular/Total cell-associated apoptotic material). (B) Proportion of internalized P . aeruginosa (Internalized/Total cell-associated bacteria). (A and B) Apoptotic cells generated by UV irradiation were stained with Annexin V-Alexa 647 and added to MDCK-Rac1-N17 filter-grown monolayers and infected with PAK-GFP. The cell-associated apoptotic material and cell-associated bacteria were quantified by image analysis as described in M&M. Dox + (control) Dox - (dominant negative Rac1). Data were normalized to control. (C) Proportion of internalized apoptotic material after pre-incubating CellTrace-labeled apoptotic cells with unlabeled Annexin V or with binding buffer alone (control) and adding them to transwell-grown lifeact-GFP MDCK monolayers. (D) Proportion of internalized P . aeruginosa after pre-incubating transwell-grown lifeact-GFP MDCK monolayers with unlabeled Annexin V for 15 min in binding buffer. (E) Transwell-grown MDCK monolayers were infected with P . aeruginosa and internalization was measured by standard antibiotic protection assays. When indicated, apoptotic cells from overgrown cultures, pre-incubated with binding buffer alone or with unlabeled-Annexin V, were added to monolayers right before infection. The mean of colony forming units (CFUs) ± SEM was calculated. Data were normalized to control. * p <0.05 vs. control, one-sample t -test.

Article Snippet: While we were preparing this manuscript, Schwarzer et al. reported that P . aeruginosa strain PA01 migrates to dying cells in scratch-wounded epithelia and that some bacteria remain immobilized on dead cells [ ].

Techniques: Bacteria, Generated, Irradiation, Staining, Infection, Control, Dominant Negative Mutation, Labeling, Binding Assay, Incubation

Journal: PLoS Pathogens

Article Title: Elimination of Pseudomonas aeruginosa through Efferocytosis upon Binding to Apoptotic Cells

doi: 10.1371/journal.ppat.1006068

Figure Lengend Snippet: (A) Projected confocal Z stack (top) and orthogonal section (bottom) showing LAMP1-positive vesicles containing bacteria. F-actin: blue, PAK-GFP: green and LAMP1: red. (B) Percentage of intracellular P . aeruginosa found within LAMP1 vesicles over time. (C) Projected confocal Z stack (top) and orthogonal section (bottom) showing acidic vesicles containing bacteria. F-actin: blue, PAK-GFP: green and LysoTracker: red. (D) Percentage of intracellular P . aeruginosa found within acidic vesicles over time. (E) Intracellular P . aeruginosa survival over time. The mean of CFUs ± SEM was calculated. Data were normalized to time 3 h. Scale bars: 5 μm.

Article Snippet: While we were preparing this manuscript, Schwarzer et al. reported that P . aeruginosa strain PA01 migrates to dying cells in scratch-wounded epithelia and that some bacteria remain immobilized on dead cells [ ].

Techniques: Bacteria