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Escherichia coli morphological changes and lipid A removal induced by reduced pressure nitrogen afterglow exposure.

Zerrouki H, Rizzati V, Bernis C, Nègre-Salvayre A, Sarrette JP, Cousty S - PLoS ONE (2015)

Bottom Line: Previous studies have demonstrated that the late afterglow region of flowing post-discharges at reduced pressure (1-20 Torr) can be used for the sterilization of surfaces and of the reusable medical instrumentation.In the present paper, we show that the antibacterial activity of a pure nitrogen afterglow can essentially be attributed to the large concentrations of nitrogen atoms present in the treatment area and not to the UV radiation of the afterglow.The afterglow exposure also results in a reduction of the lipid A proinflammatory activity, assessed by the net decrease of the redox-sensitive NFκB transcription factor nuclear translocation in murine aortic endothelial cells stimulated with control vs afterglow-treated (pure and extracted) lipid A.

View Article: PubMed Central - PubMed

Affiliation: Université de Toulouse, UPS, INPT, LAPLACE (Laboratoire Plasma et Conversion d'Energie), Bât. 3R2, F-31062, Toulouse, France; CNRS, LAPLACE, F-31062 Toulouse, France.

ABSTRACT
Lipid A is a major hydrophobic component of lipopolysaccharides (endotoxin) present in the membrane of most Gram-negative bacteria, and the major responsible for the bioactivity and toxicity of the endotoxin. Previous studies have demonstrated that the late afterglow region of flowing post-discharges at reduced pressure (1-20 Torr) can be used for the sterilization of surfaces and of the reusable medical instrumentation. In the present paper, we show that the antibacterial activity of a pure nitrogen afterglow can essentially be attributed to the large concentrations of nitrogen atoms present in the treatment area and not to the UV radiation of the afterglow. In parallel, the time variation of the inactivation efficiency quantified by the log reduction of the initial Escherichia coli (E. coli) population is correlated with morphologic changes observed on the bacteria by scanning electron microscopy (SEM) for increasing afterglow exposure times. The effect of the afterglow exposure is also studied on pure lipid A and on lipid A extracted from exposed E. coli bacteria. We report that more than 60% of lipid A (pure or bacteria-extracted) are lost with the used operating conditions (nitrogen flow QN2 = 1 standard liter per minute (slpm), pressure p = 5 Torr, microwave injected power PMW = 200 W, exposure time: 40 minutes). The afterglow exposure also results in a reduction of the lipid A proinflammatory activity, assessed by the net decrease of the redox-sensitive NFκB transcription factor nuclear translocation in murine aortic endothelial cells stimulated with control vs afterglow-treated (pure and extracted) lipid A. Altogether these results point out the ability of reduced pressure nitrogen afterglows to neutralize the cytotoxic components in Gram-negative bacteria.

No MeSH data available.


Related in: MedlinePlus

Effect of nitrogen afterglow exposure on lipid A.A. Dot blot binding assay: Increasing concentrations of lipid A were spotted on nitrocellulose membranes and blotted with an anti lipid A antibody. The relative intensity of each spot was quantified (Image J), allowing to build a dose-response calibration curve.B. Dot blots of lipid A pure (left panel) and present in E. coli extracts (right picture): 1 μg pure lipid A was spread off on sterile glass slides, and exposed to vacuum (control), or vacuum + nitrogen afterglow, in the conditions described in the Method section. At the end, the lipid A was eluted, spot on nitrocellulose membrane and immunoblotted with an anti lipid A antibody. The results are expressed as % of residual lipid A vs the vacuum-treated control. On the right panel, determination of the lipid A content in exposed bacteria. 10 μl of a bacterial solution (108/ml), were spotted on glass slides and were treated with plasma. Bacteria extracts were collected, lysed and detected by dot blot for lipid A content. Dot blot results were analyzed with the dot calibration curve and relative quantity of bacteria lipid A estimated. In insert, pictures of lipid A dot-blots pure (left) or from bacteria (right), in vacuum-treated and vacuum + nitrogen afterglow treated conditions. Mean +/-SEM of 5 separate experiments, * < p.0.05.
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pone.0116083.g004: Effect of nitrogen afterglow exposure on lipid A.A. Dot blot binding assay: Increasing concentrations of lipid A were spotted on nitrocellulose membranes and blotted with an anti lipid A antibody. The relative intensity of each spot was quantified (Image J), allowing to build a dose-response calibration curve.B. Dot blots of lipid A pure (left panel) and present in E. coli extracts (right picture): 1 μg pure lipid A was spread off on sterile glass slides, and exposed to vacuum (control), or vacuum + nitrogen afterglow, in the conditions described in the Method section. At the end, the lipid A was eluted, spot on nitrocellulose membrane and immunoblotted with an anti lipid A antibody. The results are expressed as % of residual lipid A vs the vacuum-treated control. On the right panel, determination of the lipid A content in exposed bacteria. 10 μl of a bacterial solution (108/ml), were spotted on glass slides and were treated with plasma. Bacteria extracts were collected, lysed and detected by dot blot for lipid A content. Dot blot results were analyzed with the dot calibration curve and relative quantity of bacteria lipid A estimated. In insert, pictures of lipid A dot-blots pure (left) or from bacteria (right), in vacuum-treated and vacuum + nitrogen afterglow treated conditions. Mean +/-SEM of 5 separate experiments, * < p.0.05.

Mentions: We aimed at determining whether the afterglow exposure affects the lipid A content, in the exposed bacteria. For this purpose, we developed a dot blot method based on an immuno detection of lipid A. The specific binding of primary antibody was revealed by the binding of a second antibody engineered against the first one and coupled with a horseradish peroxydase. Spots were detected using enhanced chemiluminescence reaction. We first checked the effect of nitrogen afterglow on a pure lipid A film spread on sterile glass slides (1μg), that was treated similarly to the bacteria film. The residual lipid A content was quantified using a standard lipid A curve (Fig. 4A) and compared to 1μg lipid A treated by vacuum (but not by nitrogen afterglow). As shown in Fig. 4B, the residual lipid A present on the slide after exposure to the nitrogen afterglow was less than 20% of the control, indicating that the afterglow treatment allowed to remove or degrade the endotoxin from the treated slide. In E. coli, lipid A is located in the external membrane which is the outermost layer of the Gram-negative wall which separates the external environment from the periplasm [34]. Since SEM and viability results indicate a strong alteration of the E. coli structure upon treatment with the nitrogen afterglow, we expected that this treatment should affect the lipid A content. As shown on Fig. 4, a net decrease in lipid A content was observed in bacteria treated by the nitrogen afterglow, leading to the conclusion that lipid A was submitted to afterglow transformation.


Escherichia coli morphological changes and lipid A removal induced by reduced pressure nitrogen afterglow exposure.

Zerrouki H, Rizzati V, Bernis C, Nègre-Salvayre A, Sarrette JP, Cousty S - PLoS ONE (2015)

Effect of nitrogen afterglow exposure on lipid A.A. Dot blot binding assay: Increasing concentrations of lipid A were spotted on nitrocellulose membranes and blotted with an anti lipid A antibody. The relative intensity of each spot was quantified (Image J), allowing to build a dose-response calibration curve.B. Dot blots of lipid A pure (left panel) and present in E. coli extracts (right picture): 1 μg pure lipid A was spread off on sterile glass slides, and exposed to vacuum (control), or vacuum + nitrogen afterglow, in the conditions described in the Method section. At the end, the lipid A was eluted, spot on nitrocellulose membrane and immunoblotted with an anti lipid A antibody. The results are expressed as % of residual lipid A vs the vacuum-treated control. On the right panel, determination of the lipid A content in exposed bacteria. 10 μl of a bacterial solution (108/ml), were spotted on glass slides and were treated with plasma. Bacteria extracts were collected, lysed and detected by dot blot for lipid A content. Dot blot results were analyzed with the dot calibration curve and relative quantity of bacteria lipid A estimated. In insert, pictures of lipid A dot-blots pure (left) or from bacteria (right), in vacuum-treated and vacuum + nitrogen afterglow treated conditions. Mean +/-SEM of 5 separate experiments, * < p.0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4383372&req=5

pone.0116083.g004: Effect of nitrogen afterglow exposure on lipid A.A. Dot blot binding assay: Increasing concentrations of lipid A were spotted on nitrocellulose membranes and blotted with an anti lipid A antibody. The relative intensity of each spot was quantified (Image J), allowing to build a dose-response calibration curve.B. Dot blots of lipid A pure (left panel) and present in E. coli extracts (right picture): 1 μg pure lipid A was spread off on sterile glass slides, and exposed to vacuum (control), or vacuum + nitrogen afterglow, in the conditions described in the Method section. At the end, the lipid A was eluted, spot on nitrocellulose membrane and immunoblotted with an anti lipid A antibody. The results are expressed as % of residual lipid A vs the vacuum-treated control. On the right panel, determination of the lipid A content in exposed bacteria. 10 μl of a bacterial solution (108/ml), were spotted on glass slides and were treated with plasma. Bacteria extracts were collected, lysed and detected by dot blot for lipid A content. Dot blot results were analyzed with the dot calibration curve and relative quantity of bacteria lipid A estimated. In insert, pictures of lipid A dot-blots pure (left) or from bacteria (right), in vacuum-treated and vacuum + nitrogen afterglow treated conditions. Mean +/-SEM of 5 separate experiments, * < p.0.05.
Mentions: We aimed at determining whether the afterglow exposure affects the lipid A content, in the exposed bacteria. For this purpose, we developed a dot blot method based on an immuno detection of lipid A. The specific binding of primary antibody was revealed by the binding of a second antibody engineered against the first one and coupled with a horseradish peroxydase. Spots were detected using enhanced chemiluminescence reaction. We first checked the effect of nitrogen afterglow on a pure lipid A film spread on sterile glass slides (1μg), that was treated similarly to the bacteria film. The residual lipid A content was quantified using a standard lipid A curve (Fig. 4A) and compared to 1μg lipid A treated by vacuum (but not by nitrogen afterglow). As shown in Fig. 4B, the residual lipid A present on the slide after exposure to the nitrogen afterglow was less than 20% of the control, indicating that the afterglow treatment allowed to remove or degrade the endotoxin from the treated slide. In E. coli, lipid A is located in the external membrane which is the outermost layer of the Gram-negative wall which separates the external environment from the periplasm [34]. Since SEM and viability results indicate a strong alteration of the E. coli structure upon treatment with the nitrogen afterglow, we expected that this treatment should affect the lipid A content. As shown on Fig. 4, a net decrease in lipid A content was observed in bacteria treated by the nitrogen afterglow, leading to the conclusion that lipid A was submitted to afterglow transformation.

Bottom Line: Previous studies have demonstrated that the late afterglow region of flowing post-discharges at reduced pressure (1-20 Torr) can be used for the sterilization of surfaces and of the reusable medical instrumentation.In the present paper, we show that the antibacterial activity of a pure nitrogen afterglow can essentially be attributed to the large concentrations of nitrogen atoms present in the treatment area and not to the UV radiation of the afterglow.The afterglow exposure also results in a reduction of the lipid A proinflammatory activity, assessed by the net decrease of the redox-sensitive NFκB transcription factor nuclear translocation in murine aortic endothelial cells stimulated with control vs afterglow-treated (pure and extracted) lipid A.

View Article: PubMed Central - PubMed

Affiliation: Université de Toulouse, UPS, INPT, LAPLACE (Laboratoire Plasma et Conversion d'Energie), Bât. 3R2, F-31062, Toulouse, France; CNRS, LAPLACE, F-31062 Toulouse, France.

ABSTRACT
Lipid A is a major hydrophobic component of lipopolysaccharides (endotoxin) present in the membrane of most Gram-negative bacteria, and the major responsible for the bioactivity and toxicity of the endotoxin. Previous studies have demonstrated that the late afterglow region of flowing post-discharges at reduced pressure (1-20 Torr) can be used for the sterilization of surfaces and of the reusable medical instrumentation. In the present paper, we show that the antibacterial activity of a pure nitrogen afterglow can essentially be attributed to the large concentrations of nitrogen atoms present in the treatment area and not to the UV radiation of the afterglow. In parallel, the time variation of the inactivation efficiency quantified by the log reduction of the initial Escherichia coli (E. coli) population is correlated with morphologic changes observed on the bacteria by scanning electron microscopy (SEM) for increasing afterglow exposure times. The effect of the afterglow exposure is also studied on pure lipid A and on lipid A extracted from exposed E. coli bacteria. We report that more than 60% of lipid A (pure or bacteria-extracted) are lost with the used operating conditions (nitrogen flow QN2 = 1 standard liter per minute (slpm), pressure p = 5 Torr, microwave injected power PMW = 200 W, exposure time: 40 minutes). The afterglow exposure also results in a reduction of the lipid A proinflammatory activity, assessed by the net decrease of the redox-sensitive NFκB transcription factor nuclear translocation in murine aortic endothelial cells stimulated with control vs afterglow-treated (pure and extracted) lipid A. Altogether these results point out the ability of reduced pressure nitrogen afterglows to neutralize the cytotoxic components in Gram-negative bacteria.

No MeSH data available.


Related in: MedlinePlus