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

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.