Soft nanotechnology: the potential of polyelectrolyte multilayers against E. coli adhesion to surfaces

Standard strains of Escherichia coli ATCC 35218 were obtained from the Czech Collection of Microorganisms (Brno, Czech Republic). E. coli is often used as a model organism and indicator of faecal contamination and is, therefore, included in hygiene assessment (16).

PAH (M_w=15000 g/mol, Sigma-Aldrich, St. Louis, MO, USA), (PVP-ethyl Br), and (PVP-isobutyl Br) were used as polycations, and poly(sodium 4–styrenesulfonate) (PSS; M_w=70000 g/mol, Sigma-Aldrich, USA) was used as polyanion. PVP-ethyl Br and PVP-isobutyl Br were prepared with the nucleophilic substitution of alkyl bromide on poly(4-vinylpyridine) (M_w≈60000 g/mol, Sigma-Aldrich) as described below.

PVP-ethyl Br was prepared using a modified procedure described by Okubo and Ise (17); 2.08 g of poly(4-vinylpyridine) were dissolved in 20 mL of nitromethane and added 30 mL of ethyl bromide with vigorous stirring. The mixture was heated to 45 °C and stirred overnight. The obtained precipitate was filtered, dried, and ground in a mortar. The obtained powder was solved in the filtrate, and 10 mL of ethyl bromide added. After another 24 h of heating at 45 °C, the volatiles were evaporated. The residue was dissolved in 40 mL of ethanol and added to 250 mL of dioxane. The precipitated product was filtered and dried under vacuum, yielding 3.73 g of polymer.

PVP-isobutyl Br was prepared by dissolving 3.00 g of poly(4-vinylpyridine) in 60 mL of nitromethane, and 40 mL of isobutyl bromide was added with vigorous stirring. The mixture was heated to 60 °C and stirred for seven days. During the first 24 h, the product was separated as oil. Volatiles evaporated after seven days, and the obtained residue was dissolved in 100 mL of ethanol. The precipitation of the product was induced by abrupt addition of ether (300 mL). The product was filtered, washed with ether, and dried under vacuum, yielding 4.30 g.

Polycation monomer functionalisation degrees (f) of PVP-ethyl Br and PVP-isobutyl Br were determined with potentiometric titration, either with a standardised solution of sodium hydroxide or with an AgNO₃ solution of known concentration. The monomer functionalisation degree of PSS was determined spectrophotometrically. The molar absorption coefficient of PSS(aq) in 5 % KCl(aq) by weight (e=420 L/cm/mol at 261.5 nm) used for this purpose was taken from literature (18). The following values were obtained: f (PAH)=0.89, f (PVP-ethyl)=0.91, f (PVP-isobutyl)=0.57, and f (PSS)=0.83.

Stock solutions (100 mmol/L) of polyelectrolytes were prepared by dissolving appropriate amounts of solid polyelectrolyte in miliQ water and then diluting it to 5 mmol/L or 50 mmol/L solutions. Borosilicate glass (1 mm thick, Isolab, Eschau, Germany) was cut in 1×1 cm squares, cleaned with ethanol and miliQ water, and then autoclaved before use. The glass was then coated with PEMs using a layer-by-layer method that is highly versatile for surface modification. It involves dip coating to deposit complementary molecules on the surface (14). First, the glass surface was soaked in ≈50 mL of polycation solution (PAH or PVP-ethyl Br or PVP-isobutyl Br) for five minutes and then rinsed with water. After that, it was soaked in PSS for another five minutes and rinsed with water. These two steps were repeated until five layers were deposited, and the polycation was the top layer (Figure 1).

Figure 1

E.coli from the collection were transferred to nutrient agar and incubated at 37 °C for 24 h. After that, a single colony was transferred to nutrient broth (Biolife, Milan, Italy) and incubated in the same conditions. Adhesion was tested with a modified method described by Bohinc et al. (19). Overnight E. coli culture was diluted with fresh nutrient broth in a 1:300 ratio, and the newly inoculated medium poured over the PEM-coated glass samples. Control samples were those not coated with PEMs. The samples were incubated at 37 °C for 1 h to achieve irreversible bacterial adhesion (20). After incubation, the medium was removed. Loose cells were then removed with phosphate-buffered saline (PBS) (0.026 g KH₂PO₄, 0.047 g K₂HPO₄ in 1 L) and the remaining attached bacteria stained with 0.1 % crystal violet (Merck, Darmstadt, Germany) suspension for five minutes. The dye was removed and the cells counted with a Olympus CX40 light microscope ( 400x magnification) with a CCD CMOS camera (Olympus, Tokyo, Japan). Each counting was done in triplicate and cell counts expressed as log number of cells per square millimetre (Figure 2).

Average bacterial counts (of 15 samples per PEMs or control) were compared on R software version 3.1.3 (Bell Laboratories, New Jersey, NJ, USA) with the paired Student’s t-test between control and PEM-coated surfaces. Differences were considered significant at p<0.05.