Journal: Microbiology Spectrum

Article Title: Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy

doi: 10.1128/spectrum.00191-24

Figure Lengend Snippet: NTZ drastically potentiates polymyxin B activity against E. coli . Checkerboard broth microdilution assays between nitazoxanide and polymyxin B against E. coli ATCC 25922 ( A ), colistin-resistant strain B2 ( B ), and S. Typhi Sa6 ( C ). Dark‐blue regions represent higher cell density and lower inhibition rate of combinational treatment. Data represent the mean OD (600 nm) of two biological replicates. x ‐ and y ‐axes of figures are presented as log2 scale. Synergy is defined as an FIC index of ≤0.5.

Article Snippet: In view of the close association of NuoC with oxidative phosphorylation, the red homologous recombination system was used to knock out the nuoC gene and construct the E. coli ATCC 25922 ∆ nuoC (data not shown).

Techniques: Activity Assay, Inhibition

Journal: Microbiology Spectrum

Article Title: Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy

doi: 10.1128/spectrum.00191-24

Figure Lengend Snippet: The MIC values for nitazoxanide and polymyxin B, as well as the combined effect of nitazoxanide on the MIC of polymyxin B against gram-negative strains

Article Snippet: In view of the close association of NuoC with oxidative phosphorylation, the red homologous recombination system was used to knock out the nuoC gene and construct the E. coli ATCC 25922 ∆ nuoC (data not shown).

Techniques:

Journal: Microbiology Spectrum

Article Title: Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy

doi: 10.1128/spectrum.00191-24

Figure Lengend Snippet: Time‐dependent killing of pathogens by the combination of polymyxin B and NTZ. ( A ) E. coli ATCC 25922 was grown in MHB broth then treated with PBS, polymyxin B (polymyxin, 0.125 µg/mL), or NTZ (8 µg/mL) alone or in combination (0.125 µg/mL polymyxin + 8 µg/mL NTZ). ( B ) E. coli B2 were grown in MHB broth then treated with PBS, polymyxin B (polymyxin B, 2 µg/mL), or NTZ (16 µg/mL) alone or in combination (2 µg/mL polymyxin + 16 µg/mL NTZ). The bacterial CFUs per milliliter at different time points during 24 h were determined. All experiments were performed three times, and the mean ± SD is shown. ( C ) The addition of nitazoxanide retards the evolution of polymyxin B resistance to E. coli ATCC 25922 in vitro . ( D ) Nitazoxanide and polymyxin B still have a synergistic effect (FIC ≤ 0.5) on the drug-resistant strains induced through passage.

Article Snippet: In view of the close association of NuoC with oxidative phosphorylation, the red homologous recombination system was used to knock out the nuoC gene and construct the E. coli ATCC 25922 ∆ nuoC (data not shown).

Techniques: In Vitro

Journal: Microbiology Spectrum

Article Title: Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy

doi: 10.1128/spectrum.00191-24

Figure Lengend Snippet: NTZ promotes polymyxin B to disrupt the integrity of E. coli membranes. ( A ) NTZ enhances the inhibitory effect of polymyxin B on biofilm formation. The concentrations of polymyxin B group, NTZ group, and combination group were 0.5, 32, and 0.5 µg/mL + 32 µg/mL, biofilm formation = OD drug /OD blank × 100%. Nitazoxanide enhanced the damage of polymyxin B on the outer membrane of E. coli ATCC25922 ( B ) and B2 ( D ), and promoted the fluorescence intensity of intracellular PI. ( C ) and ( E ) are histograms for quantitative fluorescence intensity analysis of PI in E. coli ATCC25922 and B2 cells, respectively. The concentrations of polymyxin B group, NTZ group, and combination group for E. coli ATCC25922 strain were 1, 128, and 1 µg/mL + 128 µg/mL, and for E. coli B strain were 8, 32, and 8 µg/mL + 32 µg/mL, respectively. ( F ) Flow cytometry assay demonstrated that NTZ enhanced the proportion of intracellular PI-stained cells in E. coli treated with polymyxin B. The concentrations of polymyxin B group, NTZ group, and combination group for E. coli ATCC25922 strain were 0.125, 16, and 0.125 µg/mL + 16 µg/mL, and for E. coli B strain were 2, 16, and 2 µg/mL + 16 µg/mL, respectively.

Article Snippet: In view of the close association of NuoC with oxidative phosphorylation, the red homologous recombination system was used to knock out the nuoC gene and construct the E. coli ATCC 25922 ∆ nuoC (data not shown).

Techniques: Membrane, Fluorescence, Flow Cytometry, Staining

Journal: Microbiology Spectrum

Article Title: Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy

doi: 10.1128/spectrum.00191-24

Figure Lengend Snippet: Morphology of E. coli ATCC 25922 was investigated by TEM after treatment of polymyxin B, NTZ alone, or the combination for 2 h, which revealed that the combination had greater damage to E. coli cells than that of high-dose polymyxin B. ( A ) CON, control group; PB, 0.5 µg/mL polymyxin; NTZ, 64 µg/mL nitazoxanide; COM, 0.5 µg/mL polymyxin B + 64 µg/mL NTZ. Note short filamentous appendages radiating from the outer membrane, thicker envelope, electron transparent, but clumped cytoplasm, mesosome. ( B ) PB 0.25, PB 0.5, PB 1, and PB 2 were treated with 0.25, 0.5, 1.0, and 2.0 µg/mL polymyxin B, respectively. The arrow points to the obvious damage site. TEM results revealed that the combination of NTZ and polymyxin B had greater damage to E. coli cells than that of high-dose polymyxin B.

Article Snippet: In view of the close association of NuoC with oxidative phosphorylation, the red homologous recombination system was used to knock out the nuoC gene and construct the E. coli ATCC 25922 ∆ nuoC (data not shown).

Techniques: Control, Membrane

Journal: Microbiology Spectrum

Article Title: Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy

doi: 10.1128/spectrum.00191-24

Figure Lengend Snippet: Synergistic mechanisms of polymyxin B–NTZ combination. ( A ) NTZ and the combination enhanced the membrane potential of E. coli ATCC 25922, as monitored by DiSC3(5). ( B ) The fluorescence intensity of the Fluo-4 AM probe observed under fluorescence microscope, and ( C ) the fluorescence intensity ratio histogram of Fluo-4 AM/DAPI; Fluo-4 AM is intracellular calcium ion concentration, DAPI is a popular chromosome counterstain. These results showed that NTZ and the combination inhibited intracellular calcium ion concentration. ( D ) Decreased production of intracellular ATP in E. coli cells treated with NTZ or the combination. ( E ) ROS production was significantly increased in E. coli cells treated with polymyxin B, NTZ, and the combination. The concentrations of polymyxin B group, NTZ group, and combination group for E. coli ATCC25922 strain were 1, 16, and 1 µg/mL + 16 µg/mL, respectively.

Article Snippet: In view of the close association of NuoC with oxidative phosphorylation, the red homologous recombination system was used to knock out the nuoC gene and construct the E. coli ATCC 25922 ∆ nuoC (data not shown).

Techniques: Membrane, Fluorescence, Microscopy, Concentration Assay

Journal: Microbiology Spectrum

Article Title: Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy

doi: 10.1128/spectrum.00191-24

Figure Lengend Snippet: Therapeutic effects of mouse peritonitis/sepsis with the combination of polymyxin B and NTZ. The combination of NTZ and polymyxin B significantly reduced bacterial load in the liver and spleen, and reduced the lesions caused by bacterial infection compared with polymyxin B alone. ( A ) Protocol of the peritonitis-sepsis model infected with E. coli ATCC 25922. Bacterial burden in the liver ( B ) and spleen ( C ) of infected mice. ( D ) Histopathological morphology of murine tissues. The liver and spleen are in order from up to down.

Article Snippet: In view of the close association of NuoC with oxidative phosphorylation, the red homologous recombination system was used to knock out the nuoC gene and construct the E. coli ATCC 25922 ∆ nuoC (data not shown).

Techniques: Infection

Journal: Microbiology Spectrum

Article Title: Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy

doi: 10.1128/spectrum.00191-24

Figure Lengend Snippet: Transcriptome analysis of E. coli ATCC 25922 after exposure to polymyxin B, nitazoxanide, or the combination of polymyxin B–nitazoxanide. ( A ) PCA showed that the clustering of internal samples of transcriptional genes in each group is very tight, while the separation between the combined group (T_5 and T_6) and other groups (C_3, C_4, T_1 and T_2) is very obvious. ( B ) Bar graph showing a large number of DEGs. The X-axis represents the different treatment groups, and the number of DEGs changes on the Y-axis. Red indicates upregulation, while green indicates downregulation. ( C ) and ( D ) are GO and KEGG enrichment analysis of DEGs of C_4 compared with T_5; ( E ) and ( F ) are GO and KEGG enrichment analysis of DEGs of T_1 compared with T_5; ( G ) and ( H ) are GO and KEGG enrichment analysis of DEGs of T_2 compared with T_5, respectively. T_1 was treated with 0.5 µg/mL polymyxin B for 2 h, T_2 was treated with 16 µg/mL nitazoxanide for 2 h, T_5 was treated with a combination of 0.5 µg/mL polymyxin B and 16 µg/mL nitazoxanide for 2 h, T_6 was treated with a combination for 0.5 h, C_3 was the blank for 0 h, and C_4 was the blank for 2 h, respectively.

Article Snippet: In view of the close association of NuoC with oxidative phosphorylation, the red homologous recombination system was used to knock out the nuoC gene and construct the E. coli ATCC 25922 ∆ nuoC (data not shown).

Techniques:

Journal: Microbiology Spectrum

Article Title: Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy

doi: 10.1128/spectrum.00191-24

Figure Lengend Snippet: The expression of oxidative phosphorylation ( A ) and flagellar assembly functional genes ( B ) was significantly inhibited by the combined treatment. The two-component system ( C ) presented a bidirectional adjustment after the combined treatment. The combined action of polymyxin B and NTZ interfered the oxidative phosphorylation and ATP synthesis, and destructed the communication network in E. coli cells.

Article Snippet: In view of the close association of NuoC with oxidative phosphorylation, the red homologous recombination system was used to knock out the nuoC gene and construct the E. coli ATCC 25922 ∆ nuoC (data not shown).

Techniques: Expressing, Phospho-proteomics, Functional Assay

Journal: Microbiology Spectrum

Article Title: Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy

doi: 10.1128/spectrum.00191-24

Figure Lengend Snippet: The strain E. coli ATCC 25922 ∆ nuoC is more sensitive to polymyxin B than ATCC 25922. ( A ) The bactericidal curve; ( B ) checkerboard broth microdilution assays between NTZ and polymyxin B against the strain E. coli ATCC 25922 ∆ nuoC (FIC = 1). Dark‐blue regions represent higher cell density and lower inhibition rate of combinational treatment. Data represent the mean OD (600 nm) of two biological replicates. x ‐ and y ‐axes of figures were presented as log2 scale. Synergy is defined as an FIC index of ≤0.5.

Article Snippet: In view of the close association of NuoC with oxidative phosphorylation, the red homologous recombination system was used to knock out the nuoC gene and construct the E. coli ATCC 25922 ∆ nuoC (data not shown).

Techniques: Inhibition

Journal: bioRxiv

Article Title: Experimental evolution of Escherichia coli on solid silver, copper, stainless steel, and glass surfaces

doi: 10.1101/2024.06.06.597739

Figure Lengend Snippet: Known and predicted interactions of the E. coli genes with relevant Gene Onthology (GO) functional annotations retrieved from EMBL STRING , default settings. Interactions of the genes belonging to the GO term “response to metal ion” (GO:0010038). Overlapping genes with the GO term “response to stress” (GO:0006950) and “stress response to metal ion (GO:0097501) are marked in red and blue, respectively. Line thickness indicates the strength of data support for the interaction.

Article Snippet: E. coli DSM 1576 (ATCC 8739, NCIB 8545, WDCM 00012, Crooks strain) was freshly ordered from the DSMZ (German Collection of Microorganisms and Cell Cultures GmbH).

Techniques: Functional Assay

Journal: bioRxiv

Article Title: Experimental evolution of Escherichia coli on solid silver, copper, stainless steel, and glass surfaces

doi: 10.1101/2024.06.06.597739

Figure Lengend Snippet: (A) and bacteriostatic effect of metal surfaces in high-organic humid (HH) conditions (B) against the ancestral E. coli ATCC 8739 strain in evolution experiment conditions. Mean values from 3 independent experiments of antibacterial activity (log10CFU/surface in inoculum – log10CFU/surface post-exposure) (A) and % growth from the inoculum (B) ±SD are presented on respective bars on graph.

Article Snippet: E. coli DSM 1576 (ATCC 8739, NCIB 8545, WDCM 00012, Crooks strain) was freshly ordered from the DSMZ (German Collection of Microorganisms and Cell Cultures GmbH).

Techniques: Activity Assay