Journal: Frontiers in Microbiology

Article Title: Antimicrobial photodynamic inactivation as an alternative approach to inhibit the growth of Cronobacter sakazakii by fine-tuning the activity of CpxRA two-component system

doi: 10.3389/fmicb.2022.1063425

Figure Lengend Snippet: Inhibitory activity of antimicrobial photodynamic inactivation on Cronobacter sakazakii . The aPDI based on hypocrellin B (A) was used to control the growth of C. sakazakii ATCC29544. The survival rate (B) of this strain was calculated relative to the dark control, according to the plate counting data. The photodynamic conditions were set as 10, 15, 20, 25, 30 μM for HB, and 20, 40, 60 J/cm 2 for light intensity, respectively. The influence of light intensity (C) and HB concentration (D) on the bacterial survival were then plotted in bar charts, whereas three control groups (blank, light only and HB only) were designed to fully indicate the aPDI effect. The representative dot plating graphs as well as the layout on the medium plate were also shown. In (C), 30 μM HB was used, and in (D) , 60 J/cm 2 light was used. To validate this effect, the LIVE/DEAD double staining was carried out (E) , and green (live), red (dead) and merged images were obtained from fluorescence microscopy. This assay was performed in cells treated with 30 μM HB. The scale bar represents 50 μm. H-L-, blank control; H+L-, single HB-treated group; H-L+, single light-treated group; H+L+, the aPDI-treated group. Values are the mean ± SEM (standard error of mean) from 6 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The harvested cells were then incubated with PI (3 μL per mL) in the dark at 37°C for 15~20 min. After washing twice with PBS to remove excess dye, the suspension was then transferred to a glass slide for fluorescence microscopy examination (ECLIPSE Ti2-U, Nikon, Tokyo, Japan) using a 590 nm bandpass filter.

Techniques: Activity Assay, Control, Concentration Assay, Double Staining, Fluorescence, Microscopy

Journal: Frontiers in Microbiology

Article Title: Antimicrobial photodynamic inactivation as an alternative approach to inhibit the growth of Cronobacter sakazakii by fine-tuning the activity of CpxRA two-component system

doi: 10.3389/fmicb.2022.1063425

Figure Lengend Snippet: Effect of aPDI on the membrane integrity of C. sakazakii . The aPDI-treated bacterial cells were subjected to PI staining. Representative fluorescence microscopy images (A) , as well as their quantification (B) were shown in cells treated with 30 μM HB photo-activated by various light doses. The scale bar represents 50 μM. In addition, DNA leakage (C) was evaluated by measuring OD 260nm of extracellular substances, and cell surface structure was profiled by scanning electron microscopy (D) . The bar represents 1 μm. Values are the mean ± SEM (standard error of mean) from 5~6 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The harvested cells were then incubated with PI (3 μL per mL) in the dark at 37°C for 15~20 min. After washing twice with PBS to remove excess dye, the suspension was then transferred to a glass slide for fluorescence microscopy examination (ECLIPSE Ti2-U, Nikon, Tokyo, Japan) using a 590 nm bandpass filter.

Techniques: Membrane, Staining, Fluorescence, Microscopy, Electron Microscopy

Journal: Frontiers in Microbiology

Article Title: Antimicrobial photodynamic inactivation as an alternative approach to inhibit the growth of Cronobacter sakazakii by fine-tuning the activity of CpxRA two-component system

doi: 10.3389/fmicb.2022.1063425

Figure Lengend Snippet: ROS stimulated by aPDI on C. sakazakii . Intracellular ROS was analyzed using DCFH-DA staining in C. sakazakii cells, in response to aPDI with 30 μM HB irradiated by various light doses. In addition to representative microscopy images (A) , fluorescence intensity (B) was quantified using Image J software. Scale bar represents 50 μm. In addition, enzymatic activity of catalase (C) was also examined to assess the bacterial antioxidative response. CAT, catalase. Values are the mean ± SEM (standard error of mean) from 3~4 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The harvested cells were then incubated with PI (3 μL per mL) in the dark at 37°C for 15~20 min. After washing twice with PBS to remove excess dye, the suspension was then transferred to a glass slide for fluorescence microscopy examination (ECLIPSE Ti2-U, Nikon, Tokyo, Japan) using a 590 nm bandpass filter.

Techniques: Staining, Irradiation, Microscopy, Fluorescence, Software, Activity Assay

Journal: Frontiers in Microbiology

Article Title: Antimicrobial photodynamic inactivation as an alternative approach to inhibit the growth of Cronobacter sakazakii by fine-tuning the activity of CpxRA two-component system

doi: 10.3389/fmicb.2022.1063425

Figure Lengend Snippet: Effect of aPDI on the CpxA-knockout mutant. ΔCpxA mutant was constructed by means of suicide plasmid-mediated recombination (A) . The survival rates (B) of wildtype and ΔCpxA were compared under the condition of 20 J/cm 2 light-30 μM HB. The representative dot-plating graphs were also shown as categorized by various dilutions (C) . Three control groups (blank, light only and HB only) were designed to indicate the aPDI effect. To validate this effect, the LIVE/DEAD double staining of both strains were performed, and green (live), red (dead) and merged images were obtained from fluorescence microscopy (D) . The scale bar represents 50 μm. The quantification was conducted using Image J software (E) . H-L-, blank control; H+L-, single HB-treated group; H-L+, single light-treated group; H+L+, the aPDI-treated group. Values are the mean ± SEM (standard error of mean) from 4~6 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The harvested cells were then incubated with PI (3 μL per mL) in the dark at 37°C for 15~20 min. After washing twice with PBS to remove excess dye, the suspension was then transferred to a glass slide for fluorescence microscopy examination (ECLIPSE Ti2-U, Nikon, Tokyo, Japan) using a 590 nm bandpass filter.

Techniques: Knock-Out, Mutagenesis, Construct, Plasmid Preparation, Control, Double Staining, Fluorescence, Microscopy, Software

Journal: Frontiers in Microbiology

Article Title: Antimicrobial photodynamic inactivation as an alternative approach to inhibit the growth of Cronobacter sakazakii by fine-tuning the activity of CpxRA two-component system

doi: 10.3389/fmicb.2022.1063425

Figure Lengend Snippet: Effect of aPDI on the membrane integrity of ΔCpxA. The aPDI-treated wildtype and ΔCpxA were subjected to PI staining. Representative fluorescence microscopy images (A) , as well as their quantification (B) were shown in cells treated with 30 μM HB photo-activated by various light doses (20 J/cm 2 , 60 J/cm 2 ). The scale bar represents 50 μM. In addition, anatomical features of C. sakazakii were observed through SEM with a scale bar set as 1 μm (C) . Values are the mean ± SEM (standard error of mean) from 4 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, p > 0.05.

Article Snippet: The harvested cells were then incubated with PI (3 μL per mL) in the dark at 37°C for 15~20 min. After washing twice with PBS to remove excess dye, the suspension was then transferred to a glass slide for fluorescence microscopy examination (ECLIPSE Ti2-U, Nikon, Tokyo, Japan) using a 590 nm bandpass filter.

Techniques: Membrane, Staining, Fluorescence, Microscopy