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

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.

Fig 1. Flowing afterglow set up.

Fig 2. Effect of nitrogen afterglow exposure on bacteria inactivation and viability.

A: Survival curves obtained for E. coli bacteria exposed to the nitrogen late afterglow with and without the MgF₂ filter. B: MTT test in bacteria control (vacuum-treated) vs bacteria exposed to the nitrogen late afterglow. Bacteria were eluted from the slides with 1 ml of Broth medium, and incubated in this medium with the MTT reagent (5 mg/ml). After 3 h incubation at 37°C, the bacteria suspension was centrifuged, and the violet formazan crystals were dissolved in 200 μl of DMSO. The optical density was estimated on a microplaque TECAN reader. C. Pictures of bacteria submitted either to vacuum alone for 15 min (LP), or to vacuum + nitrogen late afterglow (AG), and stained with the nucleic acid fluorescent probe DAPI. The results are expressed as % of the vacuum-treated control. Mean +/-SEM of 5 separate experiments, * < p.0.05.

Fig 3. Effect of nitrogen afterglow exposure on bacteria morphology.

A: Control (E. coli). B, C, D, E: After respectively 5, 10, 20 and 40 minutes of exposure to the pure N₂ afterglow.

Fig 4. 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.

Fig 5. Nuclear translocation of the NFκB transcription factor.

On the left, effect of pure lipid A: CRL2181 murine endothelial cells were treated for 20 min with lipid A (200 ng/ml) after low pressure treatment (vacuum), plasma treatment (5 Torr, 200 W, 40 min) (plasma) or from stock solution (untreated). A negative control without lipid A treatment was done (vehicle). At the end, cells were washed, the nuclei were extracted and used for SDS-PAGE electrophoresis and immunoblotting, using an anti NFκB antibody.On the right, effect of lipid A from bacteria: CRL2181 were stimulated for 2 h with 10 μl of E. coli extracts obtained after treatment with low pressure (5 Torr) for 15 min and N₂ post-discharge at 200 W for 40 min, or low pressure only, for bacteria control. The nuclei were extracted and used for immunoblotting of NFκB as for pure lipid A.