Ability of Escherichia coli to attach to plant surfaces and to compete for adhesion with the endophytic microbiota representative Alcaligenes faecalis

N. V. Limanska, I. I. Marynova, N. V. Korotaieva, A. V. Kruchanova, M. B. Galkin

Abstract


Introduction. Plants can be the possible reservoirs of Escherichia coli in nature, and it is necessary to study the possibility of these bacteria to attach to plant surfaces and compete with representatives of epiphytic and endophytic microbiota. Such studies will help to develop some biological preparations to minimize the spread of E. coli strains in environment. Taking into account the high danger of pathogenic E. coli strains, any perspective strategies for competing with E. coli on an adhesion stage should be evaluated.

Purpose. To study the ability of E. coli to attach to plant surface and the possibility to inhibit these bacteria by the antagonistic strain A. faecalis ONU 452.

Methods. To model the competition adhesion of the endophytic microbiota representative A. faecalis ONU 452 and E. coli, the strain E. coli pKEN carrying a plasmid encoding GFP-protein, has been used. Effect of E. coli pKEN GFP on plant growth was studied by inoculation of garden cress (Lepidium sativum L.) seeds with sterilized surfaces with 2% suspension of overnight culture. Germination and growth of seedlings were evaluated. Ability of E. coli pKEN to form biofilms was studied by incubation of bacterial culture with seedling roots overnight and subsequent staining of plant tissues with acridine orange (0,1%). Biofilms were observed under the light microscope (x600). Antagonistic activity of A. faecalis ONU 452 against E. coli pKEN GFP was initially found by diffusion-in-agar method. Competition for adhesion was studied under fluorescent microscope with exposition to blue range light 420 nm (x600). Cells of E. coli pKEN GFP exhibited green fluorescent opposite to non-fluorescent A. faecalis ONU 452.

Results. Growth characteristics of garden cress seedlings such as mean lengths of stems and roots increased in 30,0% when the seeds were inoculated with E. coli pKEN GFP and germinated under laboratory conditions. Percentage of germinated seeds was not changed as compared with the control. Stimulation activity was likely to be associated with the ability to attach to plant surfaces: bacteria of E. coli pKEN GFP strain formed developed biofilms on garden cress seedlings with the extensive matrix regularly covering the root surfaces. A. faecalis ONU 452 was found to be antagonistic against E. coli pKEN in a diffusion-in-agar assay. Only overnight culture inhibited E. coli pKEN GFP growth but not the filtrated cultural liquid. Due to absence of active secretion of antagonistic compounds in cultural liquid we suggested that A. faecalis ONU 452 inhibit E. coli pKEN GFP by direct cell-to-cell interactions - probably - at the stage of attachment to surfaces and biofilm formation. To test this hypothesis, we added the overnight culture of E. coli pKEN GFP diluted in different ratio to A. faecalis ONU 452 biofilms on polystyrol plates. Antagonistic strain could prevent the attachment of E. coli pKEN GFP if the ratio of A. faecalis ONU 452 : E. coli pKEN cells was 1 : 0,5 or 1 : 1. But if the number of E. coli pKEN GFP cells was higher than the concentration of antagonistic cells, E. coli could be intercalated in a formed A. faecalis ONU 452 biofilm. But as in nature the massive infiltration of certain microbial species is rarely to be occurred, it could be suggested that the present amount of antagonistic A. faecalis on plant surfaces may be sufficient for protection against E. coli penetration.

Conclusions. Bacteria of the model strain E. coli pKEN GFP attached to Lepidium sativum L. surfaces, formed developed biofilms and stimulated plant growth. Endophytic antagonistic bacterium A. faecalis ONU 452 inhibited E. coli pKEN GFP and could prevent its attachment.


Keywords


Escherichia coli; Alcaligenes faecalis; antagonist; attachment; plant surface

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