REPOSITORY > RESULTS

Abstract

The possible mechanisms for destruction of two types of bacteria with oxygen radicals were studied. Samples consisting of standard ATCC strains of Gram – positive bacteria Staphylococcus aureus and Gram – negative bacteria Escherichia coli were obtained. Bacteria were cultivated according to the standard procedures and fixed on substrates of Al foil, highly oriented graphite or highly polished silicon wafers. In last two cases, the roughness of the substrates was below a nm. The substrates with bacteria were then exposed to oxygen radicals in two different environments: 1 – inductively coupled RF plasma with the neutral atom density of 1x1022 m-3 and the ion density of 1.4x1016 m-3, and 2 – the afterglow of the oxygen plasma with the neutral atom density of 3.5x1021 m-3.
Samples were kept in plasma or its afterglow for different periods between 1 and 500 s. Immediately after the treatment, they were characterized by two complementary methods: scanning electron microscopy (SEM) and atomic force microscopy (AFM). Both techniques showed a thin film of capsule covering the bacteria as well as the space between particular bacteria in small, two – dimensional aggregates. The typical number of bacteria within an aggregate was less than 10. The SEM images obtained at low kinetic energy of primarily electrons showed that the thickness of capsule was about 100nm for S. aureus and about 150nm for E. coli.
The first effect of treatment with oxygen radicals is destruction of capsule. Both SEM and AFM images revealed that a part of capsule was disrupted in a few seconds forming numerous small droplets scattered all around bacteria. Although this effect is observed even for untreated bacteria, the number of droplets after a brief treatment increases dramatically, so it is possible to conclude that formation of numerous capsule droplets is a particular feature of treatment with oxygen radicals. The formation of the droplets was explained by a rapid increase of the surface energy of capsule due to functionalization with polar oxygen rich functional groups. The droplets, as well as the remaining capsule between particular bacteria, were completely removed with prolonged treatment.
As soon as the majority of capsule was removed, degradation of the cell wall started. AFM images showed clearly distinguishable segments on the cell walls of both types of bacteria. In any case, prolonged treatment caused increased roughness of the cell wall, tear off pieces from them until, at a certain dose, the cell wall was disrupted suddenly and these segments were scattered all around bacteria. This sudden change of the cell wall has, to the best of our knowledge, never been reported so far, and it is contradictory to classical pictures of bacterial degradation caused by plasma radicals.
After prolonged treatment the segments are almost perfectly oxidized and vanish from the surface of our samples. Once the cell wall is badly damaged, the interaction of radicals with cytoplasm starts. A slight difference in appearance between cytoplasm of E. coli and S. aureus was observed. The cytoplasm of E. coli was mainly spilled around bacteria, while the cytoplasm of S. aureus remained its spherical shape for a long time. Unlike the classical picture of the cytoplasm as a jelly-like material, our results clearly showed that the cytoplasm of S. aureus was pretty rigid since it preserved its shape even after removal of the cell wall. Our results are therefore just another confirmation of a new picture of bacterial cytoplasm. Namely, in 2001, a new theory of the cytoplasm structure was proposed by Jones et al, stating that the cytoplasm of Bacillus subtilis has a rigid structure called cytoskeleton. Our results show that the cytoplasm of S. aureus really has a well – organized structure and is not just a semi-liquid substance as claimed in classical literature.
A long treatment of bacteria with oxygen radicals causes an extensive degradation of their constituents and finally only traces of bacteria remain in the form of ash.
Knowing the flux of oxygen radicals onto the surface of bacteria, at which certain steps in the degradation occur, enables calculation of the appropriate doses of radicals. For destruction of the capsule on S. aureus the required dose is 1.6x1025m-2 and 6.6x1025m-2 for the case of plasma and afterglow treatments, respectively. These numbers are somehow lower for the case of capsule on E. coli, where the values of 0.8x1025m-2 and 1.4x1025m-2 are found. The dose required to destroy the bacterial cell wall is 7.1x1025m-2 and 1.9x1026m-2 for S. aureus placed in plasma and afterglow, respectively. The corresponding numbers for E. coli are 1.6x1025m-2 and 1x1026m-2 for plasma and afterglow, respectively. The dose required for total destruction of bacteria (only ash remains) is somehow arbitrary as it is difficult to define the term “total destruction” of bacteria. Still, in the first approximation the values are 1.9x1026m-2 and more than 2.7x1026m-2 for S. aureus in plasma and afterglow, respectively. These numbers for the case of E. coli are 9.4x1025m-2 and more than 2.7x1026m-2 for plasma and afterglow, respectively.
According to our experimental results, a new model of three – dimensional organization of peptidogycan in E. coli cell wall is proposed. At this model, peptidoglycan is organized in flat, oval or round shaped pieces composed in such a manner, where pieces overlapping each other and looks like fish scales or tiles on a roof.