Journal: Bio-protocol

Article Title: Framework for Analyzing the Anti-biofilm and Anti-virulence Activities of Fatty Acids from Hermetia illucens Larvae Targeting Multidrug-Resistant Klebsiella pneumoniae

doi: 10.21769/BioProtoc.5629

Figure Lengend Snippet: The 96-well microplate is filled with 100 μL of LB broth in columns 1–5 and 7–12. Subsequently, 100 μL of the AWME3 extract is added and serially diluted across columns 1–4 and 7–10. Next, 100 μL of the corresponding bacterial suspension is added to each well, with the exception of column 12, which is the blank. After incubation for 4 h, membrane permeability is evaluated by adding 10 μL of crystal violet solution to each well. The absorbance of the supernatant is measured at OD 570 nm after incubation in the dark for 15 min. All experimental conditions are performed in triplicate. Columns 5 and 11 serve as untreated controls, while column 12 (triplicate) is used as the blank. Numbers in the rows correspond to the tested K. pneumoniae strains: (1) KPi1627, (2) KPM9, and (3) K. pneumoniae ATCC BAA-2473.

Article Snippet: Grow K. pneumoniae ATCC BAA-2473 strain to the mid-logarithmic phase, corresponding to an optical density (OD 600 ) of 0.5 ± 0.05.

Techniques: Suspension, Incubation, Membrane, Permeability

Journal: Bio-protocol

Article Title: Framework for Analyzing the Anti-biofilm and Anti-virulence Activities of Fatty Acids from Hermetia illucens Larvae Targeting Multidrug-Resistant Klebsiella pneumoniae

doi: 10.21769/BioProtoc.5629

Figure Lengend Snippet: (A, B) Kpi1627, KPM9, and KP ATCC BAA-2473 strains were cultured in LB broth and incubated overnight at 37 °C. Then, all strains were treated with 0.5 MIC (0.125 mg/mL) of AWME3, which did not show any turbidity and mucoviscosity. (C) Centrifugation for 10 min at low speed (1,000× g ) led to high precipitation of treated and untreated strains, and dense pellets were formed.

Article Snippet: Grow K. pneumoniae ATCC BAA-2473 strain to the mid-logarithmic phase, corresponding to an optical density (OD 600 ) of 0.5 ± 0.05.

Techniques: Cell Culture, Incubation, Centrifugation

Journal: Bio-protocol

Article Title: Framework for Analyzing the Anti-biofilm and Anti-virulence Activities of Fatty Acids from Hermetia illucens Larvae Targeting Multidrug-Resistant Klebsiella pneumoniae

doi: 10.21769/BioProtoc.5629

Figure Lengend Snippet: Column 10 represents the control group, while column 11 is the blank. 100 μL of LB broth is added to columns 1–8, and 100 μL of AWME3 extract at various final concentrations (0.08–1.0 mg/mL) is added and serially diluted across columns 1–8. Then, 100 μL bacterial suspension is added to each well (except column 11). After incubation for 24 h without shaking, biofilm disruption is evaluated by adding 125 μL of 1.5% CV solution to each well, followed by 125 μL of acetic acid. Plates are then incubated for 10 min at room temperature. Absorbance is measured at OD 550 nm. All experimental conditions were performed in triplicate. Numbers in the rows correspond to the tested K. pneumoniae strains: (1) Kpi1627 and (2) ATCC BAA-2473.

Article Snippet: Grow K. pneumoniae ATCC BAA-2473 strain to the mid-logarithmic phase, corresponding to an optical density (OD 600 ) of 0.5 ± 0.05.

Techniques: Control, Suspension, Incubation, Disruption

Journal: Bio-protocol

Article Title: Framework for Analyzing the Anti-biofilm and Anti-virulence Activities of Fatty Acids from Hermetia illucens Larvae Targeting Multidrug-Resistant Klebsiella pneumoniae

doi: 10.21769/BioProtoc.5629

Figure Lengend Snippet: (A–D) Fluorescence microscopy representative images of (A) untreated cells (control) biofilm established by Kp ATCC BAA-2473 strain, and the same bacteria treated with (B) 0.5 MIBC (0.25 mg/mL), (C) MIBC (0.5 mg/mL), and (D) 2 MIC (1 mg/mL) of AWME3. PI staining was used to stain eNAs. (E) Relative fluorescence intensity of biofilm structures of the Kp ATCC BAA-2473 strain is reported in arbitrary units (a.u) obtained after quantification of digital images using Fiji ImageJ software. Data are expressed as the mean ± STD. Statistical significance was calculated using ordinary one-way ANOVA with Dunnett’s multiple comparison test (****p < 0.0001).

Article Snippet: Grow K. pneumoniae ATCC BAA-2473 strain to the mid-logarithmic phase, corresponding to an optical density (OD 600 ) of 0.5 ± 0.05.

Techniques: Fluorescence, Microscopy, Control, Bacteria, Staining, Software, Comparison

Journal: Bio-protocol

Article Title: Framework for Analyzing the Anti-biofilm and Anti-virulence Activities of Fatty Acids from Hermetia illucens Larvae Targeting Multidrug-Resistant Klebsiella pneumoniae

doi: 10.21769/BioProtoc.5629

Figure Lengend Snippet: SEM micrographs of K. pneumoniae ATCC BAA-2473 biofilms were developed on a glass slide surface and incubated for 24 h at 37 °C. (A–C) Untreated biofilm at different magnifications. (D–F) Treated biofilms with (D) 0.5 MIBC (0.25 mg/mL), (E) MIBC (0.5 mg/mL), and (F) 2 MIBC (1 mg/mL) of AWME3. All figures show SEM images with a magnification range from 5,490× to 55,700×. Blue arrows refer to (D) membrane disruption and cell wall degradation or (E) cell wall rupture and severe degradation, and the red cycle refers to bacterial debris.

Article Snippet: Grow K. pneumoniae ATCC BAA-2473 strain to the mid-logarithmic phase, corresponding to an optical density (OD 600 ) of 0.5 ± 0.05.

Techniques: Incubation, Membrane, Disruption

Journal: Bio-protocol

Article Title: Framework for Analyzing the Anti-biofilm and Anti-virulence Activities of Fatty Acids from Hermetia illucens Larvae Targeting Multidrug-Resistant Klebsiella pneumoniae

doi: 10.21769/BioProtoc.5629

Figure Lengend Snippet: The 96-well microplate is filled with 100 μL of LB broth in columns 1–5 and 7–12. Subsequently, 100 μL of the AWME3 extract is added and serially diluted across columns 1–4 and 7–10. Next, 100 μL of the corresponding bacterial suspension is added to each well, with the exception of column 12, which is the blank. After incubation for 4 h, membrane permeability is evaluated by adding 10 μL of crystal violet solution to each well. The absorbance of the supernatant is measured at OD 570 nm after incubation in the dark for 15 min. All experimental conditions are performed in triplicate. Columns 5 and 11 serve as untreated controls, while column 12 (triplicate) is used as the blank. Numbers in the rows correspond to the tested K. pneumoniae strains: (1) KPi1627, (2) KPM9, and (3) K. pneumoniae ATCC BAA-2473.

Article Snippet: This protocol outlines a step-by-step method for extracting and analyzing the anti-biofilm and anti-virulence properties of AWME3 on three K. pneumoniae strains: KPM9 (mucoviscous), KPi1627 (hyper-mucoviscous), and K. pneumoniae ATCC BAA-2473 (non-mucoid, carbapenem-resistant).

Techniques: Suspension, Incubation, Membrane, Permeability

Journal: Bio-protocol

Article Title: Framework for Analyzing the Anti-biofilm and Anti-virulence Activities of Fatty Acids from Hermetia illucens Larvae Targeting Multidrug-Resistant Klebsiella pneumoniae

doi: 10.21769/BioProtoc.5629

Figure Lengend Snippet: (A, B) Kpi1627, KPM9, and KP ATCC BAA-2473 strains were cultured in LB broth and incubated overnight at 37 °C. Then, all strains were treated with 0.5 MIC (0.125 mg/mL) of AWME3, which did not show any turbidity and mucoviscosity. (C) Centrifugation for 10 min at low speed (1,000× g ) led to high precipitation of treated and untreated strains, and dense pellets were formed.

Article Snippet: This protocol outlines a step-by-step method for extracting and analyzing the anti-biofilm and anti-virulence properties of AWME3 on three K. pneumoniae strains: KPM9 (mucoviscous), KPi1627 (hyper-mucoviscous), and K. pneumoniae ATCC BAA-2473 (non-mucoid, carbapenem-resistant).

Techniques: Cell Culture, Incubation, Centrifugation

Journal: Bio-protocol

Article Title: Framework for Analyzing the Anti-biofilm and Anti-virulence Activities of Fatty Acids from Hermetia illucens Larvae Targeting Multidrug-Resistant Klebsiella pneumoniae

doi: 10.21769/BioProtoc.5629

Figure Lengend Snippet: Column 10 represents the control group, while column 11 is the blank. 100 μL of LB broth is added to columns 1–8, and 100 μL of AWME3 extract at various final concentrations (0.08–1.0 mg/mL) is added and serially diluted across columns 1–8. Then, 100 μL bacterial suspension is added to each well (except column 11). After incubation for 24 h without shaking, biofilm disruption is evaluated by adding 125 μL of 1.5% CV solution to each well, followed by 125 μL of acetic acid. Plates are then incubated for 10 min at room temperature. Absorbance is measured at OD 550 nm. All experimental conditions were performed in triplicate. Numbers in the rows correspond to the tested K. pneumoniae strains: (1) Kpi1627 and (2) ATCC BAA-2473.

Article Snippet: This protocol outlines a step-by-step method for extracting and analyzing the anti-biofilm and anti-virulence properties of AWME3 on three K. pneumoniae strains: KPM9 (mucoviscous), KPi1627 (hyper-mucoviscous), and K. pneumoniae ATCC BAA-2473 (non-mucoid, carbapenem-resistant).

Techniques: Control, Suspension, Incubation, Disruption

Journal: Bio-protocol

Article Title: Framework for Analyzing the Anti-biofilm and Anti-virulence Activities of Fatty Acids from Hermetia illucens Larvae Targeting Multidrug-Resistant Klebsiella pneumoniae

doi: 10.21769/BioProtoc.5629

Figure Lengend Snippet: (A–D) Fluorescence microscopy representative images of (A) untreated cells (control) biofilm established by Kp ATCC BAA-2473 strain, and the same bacteria treated with (B) 0.5 MIBC (0.25 mg/mL), (C) MIBC (0.5 mg/mL), and (D) 2 MIC (1 mg/mL) of AWME3. PI staining was used to stain eNAs. (E) Relative fluorescence intensity of biofilm structures of the Kp ATCC BAA-2473 strain is reported in arbitrary units (a.u) obtained after quantification of digital images using Fiji ImageJ software. Data are expressed as the mean ± STD. Statistical significance was calculated using ordinary one-way ANOVA with Dunnett’s multiple comparison test (****p < 0.0001).

Article Snippet: This protocol outlines a step-by-step method for extracting and analyzing the anti-biofilm and anti-virulence properties of AWME3 on three K. pneumoniae strains: KPM9 (mucoviscous), KPi1627 (hyper-mucoviscous), and K. pneumoniae ATCC BAA-2473 (non-mucoid, carbapenem-resistant).

Techniques: Fluorescence, Microscopy, Control, Bacteria, Staining, Software, Comparison

Journal: Bio-protocol

Article Title: Framework for Analyzing the Anti-biofilm and Anti-virulence Activities of Fatty Acids from Hermetia illucens Larvae Targeting Multidrug-Resistant Klebsiella pneumoniae

doi: 10.21769/BioProtoc.5629

Figure Lengend Snippet: SEM micrographs of K. pneumoniae ATCC BAA-2473 biofilms were developed on a glass slide surface and incubated for 24 h at 37 °C. (A–C) Untreated biofilm at different magnifications. (D–F) Treated biofilms with (D) 0.5 MIBC (0.25 mg/mL), (E) MIBC (0.5 mg/mL), and (F) 2 MIBC (1 mg/mL) of AWME3. All figures show SEM images with a magnification range from 5,490× to 55,700×. Blue arrows refer to (D) membrane disruption and cell wall degradation or (E) cell wall rupture and severe degradation, and the red cycle refers to bacterial debris.

Article Snippet: This protocol outlines a step-by-step method for extracting and analyzing the anti-biofilm and anti-virulence properties of AWME3 on three K. pneumoniae strains: KPM9 (mucoviscous), KPi1627 (hyper-mucoviscous), and K. pneumoniae ATCC BAA-2473 (non-mucoid, carbapenem-resistant).

Techniques: Incubation, Membrane, Disruption