Journal: International Journal of Nanomedicine

Article Title: Graphene oxide/multi-walled carbon nanotubes as nanofeatured scaffolds for the assisted deposition of nanohydroxyapatite: characterization and biological evaluation

doi: 10.2147/IJN.S106339

Figure Lengend Snippet: Bactericidal effect of nHAp/MWCNT-GO nanocomposites. Notes: Bactericidal effect against ( A ) Escherichia coli and ( B ) Staphylococcus aureus . Values are reported as mean ± SD (n=3). * P <0.05. Abbreviations: SD, standard deviation; MWCNT, multi-walled carbon nanotube; GO, graphene oxide; nHAp, nanohydroxyapatite.

Article Snippet: To evaluate the bacterial effect of samples, Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) strains were used as Gram-negative and Gram-positive bacteria models, respectively.

Techniques: Standard Deviation

Journal: Microbiology (Reading, England)

Article Title: Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death.

doi: 10.1099/mic.0.2007/007427-0

Figure Lengend Snippet: Fig. 1. Intracellular type III secretion of S. flexneri in infected macrophages. J774 macrophages were infected with GFP- expressing S. flexneri wild-type M90T or the secretion-deficient mutant strain DmxiD at an m.o.i. of 20 for 75 min, and at the time points indicated immunofluorescence microscopy was used to visualize (A) the secretion of IpaB or (B) the IpaB pool within S. flexneri. Bars, 2 mm. To stain IpaB in the bacterial cytosol (B, C), S. flexneri was permeabilized with lysozyme before the immuno-staining with monoclonal IpaB H16 and Cy3-conjugated goat anti-mouse antibodies (red). (C) The amount of cytosolic and surface-bound IpaB present prior to the infection was analysed using exponentially growing wild-type S. flexneri immobilized on gelatin-coated coverslips. Bars, 3 mm. Images are representative of at least three independent experiments.

Article Snippet: Samples were incubated for 1 h with either a monoclonal mouse anti-IpaB primary antibody (clone H16, 1 : 300; Barzu et al., 1993) or a monoclonal rat anti-LAMP-1 antibody (clone 1D4B, 1 : 50; Developmental Studies Hybridoma Bank, University of Iowa), followed by Cy3-conjugated goat anti-mouse (1 : 200; Jackson Immuno Research Laboratories) or TRITC-labelled rabbit anti-rat IgG (1 : 200; Sigma) secondary antibodies (1 h).

Techniques: Infection, Expressing, Mutagenesis, Immunofluorescence, Microscopy, Staining, Immunostaining

Journal: Microbiology (Reading, England)

Article Title: Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death.

doi: 10.1099/mic.0.2007/007427-0

Figure Lengend Snippet: Fig. 2. The protonophore CCCP inhibits type III secretion of S. flexneri triggered by Congo red or in macrophages. (A) Type III secretion of S. flexneri wild-type (M90T) was induced with the dye Congo red in vitro after treatment with CCCP at the concentrations indicated, and IpaB in the bacterial supernatant or pellets was determined by Western blotting. (B) Immunofluorescence micrographs of J774 macrophages infected for 45 min with GFP-expressing wild-type S. flexneri (m.o.i. 20) in the presence or absence of 25 or 100 mM CCCP added 15 min post-infection. Bar, 5 mm. (C) J774 macrophages were infected (m.o.i. 100) with S. flexneri wild-type (M90T) or DmxiD in the presence or absence of 25 mM CCCP added 15 min post-infection. At 45 min post-infection, lysates of the infected macrophages were fractionated by centrifugation into bacteria/ macrophage debris (‘Intracellular bacteria’) or macrophage (MW) cytosol and membranes, respectively. S. flexneri IpaB and the endoplasmic reticulum membrane protein calnexin were detected by Western blotting. Similar results were obtained in four (A, C) or three (B) independent experiments.

Article Snippet: Samples were incubated for 1 h with either a monoclonal mouse anti-IpaB primary antibody (clone H16, 1 : 300; Barzu et al., 1993) or a monoclonal rat anti-LAMP-1 antibody (clone 1D4B, 1 : 50; Developmental Studies Hybridoma Bank, University of Iowa), followed by Cy3-conjugated goat anti-mouse (1 : 200; Jackson Immuno Research Laboratories) or TRITC-labelled rabbit anti-rat IgG (1 : 200; Sigma) secondary antibodies (1 h).

Techniques: In Vitro, Western Blot, Immunofluorescence, Infection, Expressing, Centrifugation, Bacteria, Membrane

Journal: Microbiology (Reading, England)

Article Title: Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death.

doi: 10.1099/mic.0.2007/007427-0

Figure Lengend Snippet: Fig. 7. Expression of IpaB from a plasmid increases cytotoxicity of wild-type S. flexneri. (A) J774 macrophages were infected with wild-type S. flexneri M90T, the non-polar ipaB deletion mutant DipaB or the secretion-deficient mutant DmxiD (m.o.i. 50) carrying the arabinose-inducible IpaB expression plasmid pGNS025 where indicated. The strains were grown without arabinose (white bars), or in the presence of 0.1 % arabinose added to the subculture prior to (hatched white bars) or concomitant with (black bars) an infection . Cytotoxicity was measured by LDH release 2 h 45 min post-infection. The data shown are means and standard deviation of triplicates. (B) To control expression and secretion of IpaB produced by pGNS025, subcultures of the S. flexneri strains M90T, DipaB or DmxiD carrying the IpaB expression plasmid were grown in the presence or absence of 0.1 % arabinose. Type III secretion was induced with the dye Congo red, and the amount of IpaB in the supernatant or in bacterial pellets was determined by Western blotting. Similar results were obtained in two (A) or four (B) independent experiments.

Article Snippet: Samples were incubated for 1 h with either a monoclonal mouse anti-IpaB primary antibody (clone H16, 1 : 300; Barzu et al., 1993) or a monoclonal rat anti-LAMP-1 antibody (clone 1D4B, 1 : 50; Developmental Studies Hybridoma Bank, University of Iowa), followed by Cy3-conjugated goat anti-mouse (1 : 200; Jackson Immuno Research Laboratories) or TRITC-labelled rabbit anti-rat IgG (1 : 200; Sigma) secondary antibodies (1 h).

Techniques: Expressing, Plasmid Preparation, Infection, Mutagenesis, Standard Deviation, Control, Produced, Western Blot

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: Precision targeting of H. pylori using probiotic delivery of guided antimicrobial peptides (gAMPs). The probiotic Lactococcus lactis carries the Escherichia coli / L. lactis shuttle vector, pTKR, for the expression of gAMPs in the mouse stomach, allowing rapid engineering in E. coli and transfer to L. lactis . The gAMPs were placed downstream of the acid-inducible P1 promoter and the usp secretion signal peptide to allow secretion in the acidic stomach environment. The guide attached to the N-terminus of the AMP was that portion of the human thrombin protein, Multimerin-1, that binds to H. pylori .

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: Plasmid Preparation, Expressing

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: MM1-guided GFP (MM1-GFP) protein binds specifically to H. pylori cells. ( A ) Protein preparations of MM1-GFP, but not GFP, bound to H. pylori cells. Fluorescence intensity of cells of target bacterium H. pylori 60,190 WT untreated (red) or treated with GFP (green) or MM1-GFP (blue) with averaged median fluorescence ( n = 3) in relative fluorescence units (RFUs) obtained from BD FACSverse flow cytometer using blue 488 nm laser and a 488/10 bandpass filter; standard deviation shown; statistical significance (one-way analysis of variance, one-way ANOVA; ns, not significant; *** P ≤ 0.001). (B and C) Neither MM1-GFP nor GFP protein significantly bound to off-target bacterial cells. Flow cytometry as in ( A ) for the cells of off-target bacteria Lactobacillus plantarum ( B ) or Escherichia coli K12 ( C ). ( D ) Confocal microscopy demonstrated that MM1-GFP, but not GFP, bound strongly to Helicobacter pylori cells. Imaging of H. pylori 60,190 WT cells untreated (first column) or treated with GFP (second column) or MM1-GFP (third column). Top row: visualization of GFP or MM1-GFP fluorescence at 488 nm. Middle row: visualization of bacterial cells (CellBrite stain, 640 nm). Bottom row: merged images.

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: Fluorescence, Flow Cytometry, Standard Deviation, Bacteria, Confocal Microscopy, Imaging, Staining

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: gAMP probiotics selectively kill H. pylori when co-cultured in vitro . Guided (red) or unmodified (blue) versions of three AMPs (alyteserin, CRAMP, and laterosporulin) were expressed by engineered L. lactis, which was co-cultured with the target ( H. pylori ) or the non-target bacteria, E. coli and Lactobacillus . Eight different initial probiotic concentrations were tested for each AMP or gAMP (x-axis), and the titer of the H. pylori or off-target bacterium was measured after 24 hours of co-culture (y-axis). Titers were determined starting with qPCR using vacA primers for H. pylori , DE3-T7 polymerase primers for E. coli, recA primers for Lactobacillus , and acma primers for L. lactis . The corresponding CFU values were calculated from standard curves of C T versus CFU using CFU values obtained by bacterial dilution and plating . The limits of qPCR detection differed between bacterial species resulting in flat-lining at different low-end levels.

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: Probiotics, Cell Culture, In Vitro, Bacteria, Co-Culture Assay

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: gAMP and AMP probiotics control H. pylori in the mouse model both as a therapeutic and a prophylactic. ( A ) For both therapeutic and prophylactic experiments, the H. pylori infection was established by oral gavage for four consecutive days with 250 µL of resuspended H. pylori (~5 × 10 7 CFU/mL). For therapeutic experiments, the infection was followed by a dose of 250 µL of resuspended L. lactis (~5 × 10 7 CFU/mL) on day 5 of the regimen. For prophylactic experiments, the probiotic was provided on day 0, followed by a H. pylori challenge on days 3–6. Immediately before administration of both H. pylori and L. lactis , mice stomach samples were extracted by reverse oral gavage method. Further samples were extracted on day 8 and 10 by the same method. For each treatment listed in ( B ) and ( C ), at least six mice were used. ( B ) H. pylori titer measured over the time course of the therapeutic experiment. On each day, probiotics expressing gAMP or AMP were compared to antibiotic treatment or negative controls. H. pylori titers in the reverse oral gavage stomach samples were determined by qPCR using the CFU vs C T standard curve for H. pylori . The strength of infection is color-coded. Complete data with significance values are presented in . ( C ) H. pylori titer measured over the time course of the prophylactic experiment. The same probiotic and negative control treatments and H. pylori titer determinations were used as in ( B ).

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: Probiotics, Control, Infection, Expressing, Negative Control

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: Probiotic gAMP/AMP treatment in vivo reverses the degradation of taxonomic richness caused by H. pylori infection. ( A and C ) The left two panels cover the therapeutic experiment, displaying the taxonomic analyses of the samples from . ( B and D ) The right two panels cover the prophylactic experiment, with taxonomic analyses of the samples of . ( A and B ) The top panels display the major genera found on each sampling day for each treatment, with H. pylori infection leading to domination by Acinetobacter and Staphylococcus and a relief from this domination being provided by the probiotic treatments. ( C and D ) The bottom panels chart the taxonomic richness by a simple ASV log 10 count over time for each treatment group, with a rebound or retention of taxonomic richness in the probiotic treatment group, especially the gAMP probiotic group.

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: In Vivo, Infection, Sampling

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: The Microbial Dysbiosis Index (MDI) comprised 10 co-varying taxa that also correlated with H. pylori infection. ( A ) Compositional Correlation network of 10 genera revealed by the CCREPE analysis, whose composition co-varied among the mice stomach samples on days 0 and 5, which were pre- and post -H. pylori infection, respectively. Eight of these were positively correlated with each other (blue lines), while two others ( Staphylococcus and Acinetobacter ) were positively correlated with each other but inversely correlated with the other 8 (red lines). All the correlations were significant ( P < 0.05, q < 0.10). ( B ) The 10 genera that co-varied significantly also had a significant change in their mean relative abundance among the samples of day 0 vs day 5, with 8 of them decreasing following H. pylori infection while Staphylococcus and Acinetobacter saw a significant increase. This further validates the importance of these genera as markers for mice stomach microbial health in our experiment. ( C ) Principal Components Analysis (PCA) of the taxonomic relationship between the microbiota of all 350 samples in the study. Dysbiotic samples (red) clustered separately from non-dysbiotic samples (blue), demonstrating that MDI correlated with the taxonomic relatedness of the samples generally. Jitter was used to allow all samples to be in view. ( D ) The same PCA as in ( C ) but with ordinates overlayed corresponding to the 10 genera of the MDI, with longer vectors indicating more correspondence to the abundance of that genus. Staphylococcus and Acinetobacter are again seen as strong predictors for H. pylori /no treatment samples (red). No jitter was used in order to report the unaltered PCA output.

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: Infection

Journal: Microbiology Spectrum

Article Title: Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides

doi: 10.1128/spectrum.02014-23

Figure Lengend Snippet: Probiotic gAMP treatment protects against microbial dysbiosis. ( A ) At the start of the therapeutic experiment (day 0), the MDIs of all samples (dark blue) were negative (healthy) and had predominantly high taxonomic richness (circle size). After H. pylori infection and probiotic treatment (days 8 and 10 combined), untreated (“only H. pylori ”) and empty probiotic-treated samples were dysbiotic with low taxonomic richness, while AMP and gAMP probiotic-treated samples were healthy and had high taxonomic richness. Antibiotic treatment relieved dysbiosis but had low taxonomic richness. ( B ) In the prophylactic experiment, all probiotic treatments protected against H. pylori -induced dysbiosis, with gAMP probiotics promoting the most robust taxonomic richness. Infected mice without the prophylactic yielded only dysbiotic samples. ( C ) Dysbiosis in the therapeutic experiment, tracked by day. All treatments showed a negative (healthy) MDI for day 0 and a positive (dysbiotic) MDI for day 5 since these samples were collected just before oral inoculation with H. pylori or probiotic, respectively. The bar colors correspond to the colors and treatments in ( A ). Within 3 days, dysbiosis had been alleviated by gAMP and AMP probiotics and antibiotics but not by the empty vector probiotic. ( D ) Dysbiosis in the prophylactic experiment, tracked by day. All samples had healthy MDI values before (day 0) and 3 days after probiotic treatment, but H. pylori induced dysbiosis by days 8 and 10 in samples lacking prophylaxis (null treatment, magenta). All probiotic treatments protected against H. pylori -induced dysbiosis.

Article Snippet: E. coli 10β (DH10B derivative) was purchased from New England Biolabs (NEB #C3019I) and was grown in Luira-Betani (LB) broth/agar (Thermo Scientific) at 37°C with aeration.

Techniques: Infection, Probiotics, Plasmid Preparation

Journal: PLoS ONE

Article Title: Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic Escherichia coli

doi: 10.1371/journal.pone.0101924

Figure Lengend Snippet: Representative whole cell MALDI-TOF mass spectrum of the Shiga-Toxigenic E. coli outbreak isolate TY-2482 acquired after formic acid extraction. Inlays show enlarged views of outbreak strain specific marker peaks and the amino acid sequence of the corresponding proteins. Peptides identified by LC-MS/MS are indicated by a gray background. The tick mark interval in the enlarged peak views is set to 100.

Article Snippet: In addition to the samples, preparations of a mixture of E. coli strain DH5α proteins (Bacterial Protein Standard, Bruker Daltonics) were spotted on each target for instrument calibration.

Techniques: Extraction, Marker, Sequencing, Liquid Chromatography with Mass Spectroscopy

Journal: PLoS ONE

Article Title: Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic Escherichia coli

doi: 10.1371/journal.pone.0101924

Figure Lengend Snippet: Mean signal to noise ratio of outbreak strain marker peaks (A, B) and mean signal intensitiy (C, D) at marker peak position in formic acid extraction (A, C) and direct sample deposition (B, D) triplicate spectra from 293 study isolates. Black triangles and white circles represent measurements from 104 outbreak and 189 non outbreak E. coli isolates, respectively. Red colour indicates misidentified isolates.

Article Snippet: In addition to the samples, preparations of a mixture of E. coli strain DH5α proteins (Bacterial Protein Standard, Bruker Daltonics) were spotted on each target for instrument calibration.

Techniques: Marker, Extraction

Journal: Journal of the Association of Medical Microbiology and Infectious Disease Canada

Article Title: 2023 Annual Conference Conférence Annuelle

doi: 10.3138/jammi.8.s1.abst

Figure Lengend Snippet: Weighted proportion of antibiogram and % susceptibility.

Article Snippet: Antibiotic susceptibility of bacterial isolates of blood and urine samples from 2013 to 2021 of a tertiary-care academic hospital was obtained from annual antibiograms created following the Clinical and Laboratory Standards Institute (CLSI) M39 document.

Techniques:

Journal: NPJ Biofilms and Microbiomes

Article Title: Ugly ducklings—the dark side of plastic materials in contact with potable water

doi: 10.1038/s41522-018-0050-9

Figure Lengend Snippet: Number of bacteria in biofilms from the inner surface of bath toys. Flow cytometry was used to determine the number of bacterial cells in bath toy biofilms using SYBR ® Green I staining following biofilm removal and dispersal. Bath toys were either from real households (real bath toys), or used under controlled conditions with clean water prior to bathing (clean water controls) or with used water after bathing (dirty water controls). Error bars represent standard deviation of triplicate measurements

Article Snippet: Five hundred microliters of each sample were either stained with 5 μL SYBR ® Green I (SG, Invitrogen AG, Basel, Switzerland; 100× diluted in Tris buffer, pH 8) to detect the TCC or with 5 μL SG with additional propidium iodide (final concentration of 0.3 mM) to quantify the intact cell concentration.

Techniques: Bacteria, Flow Cytometry, SYBR Green Assay, Staining, Standard Deviation