Bacterial attachment to surfaces and proliferation under favourable conditions can lead to biofilm growth, lower product quality, a failed industrial process, and eventually adverse health issues (1). This bacterial surface attachment depends on several factors, including bacterial cell properties (surface charge, flagella, and extracellular polymeric substances), surface properties (roughness, surface charge, and chemical composition), and environmental conditions (nutrient, temperature, pH, and the presence of antimicrobial substances) (2). During the initial stage of cell adhesion, bacterial cells interact with substrate surface chemically and physically. Interactions typically contributing to bacterial adhesion are electrostatic, van der Waals, and hydrophobic or hydrophilic (3).
The most effective approach to bacterial management is to prevent cell adhesion rather than to treat it. Some have tried to accomplish this with antimicrobial coating and others by modifying surface properties (4). The anti-adhesive effect can be achieved by inhibiting close contact between the cell and the surface with hydrophilic polymer-based polyelectrolyte multilayers (PEMs) or by stiffening the surface so that the cells cannot attach (5).
The last decade saw a remarkable progress with PEMs (6, 7, 8, 9), which have good antibacterial properties but are not toxic to eukaryotes, humans included. Another appealing advantage of PEMs is that their layered structures can be tuned with nanoscale precision to obtain desired surface properties (9). PEMs owe their antibacterial effects to hydrophobicity and charge interaction, which destabilises and disrupts bacterial cells (10, 11, 12, 13, 14, 15).
The aim of this study was to compare the anti-adhesion potential of newly synthesised poly(4-vinyl-
Standard strains of
PAH (
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 (
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).
Sample coating and anti-adhesion assessment
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
Surfaces coated with PAH/PSS PEMs showed significantly lower bacterial counts than uncoated control glass surfaces (p<0.05). Moreover, bacterial counts decreased with higher polyelectrolyte concentrations (50 mmol/L). The best anti-adherent effect was achieved with PVP-ethyl Br/PSS at 50 mmol/L, which reduced bacterial adhesion up to 60 %, followed by PVP-isobutyl Br/PSS (38.4 %), and PAH/PSS (38.1 %) (Table 1).
Light microscopy confirmed significant reduction in
As expected, our findings have confirmed anti-adhesion properties of polyanion top layers in PEMs (22–25). Kovačić et al. (23), in fact, reported that only 20 % of
Our study has also demonstrated the efficacy of polycation top layer against bacterial adhesion. Guo et al. (8) reported that polycations with quaternary ammonium groups have sufficient charge density on flexible backbones to prevent adhesion. The interaction between positively charged molecules and negatively charged cell membranes can cause leakage of intracellular constituents and, consequently, cell detachment.
Besides concentration, the structure of polyelectrolytes seems to have a significant role in antibacterial properties of PEMs. We observed that polycations with quaternary amine groups (PVP-ethyl and PVP-isobutyl) had greater anti-adhesive potential than polycations with primary amine groups (PAH). This may be related to different charge density of the polyion, which in PAH depends on pH. Furthermore, some steric factors and hydrophobicity of the monomers could have influenced the observed behaviour of PEMs. It is expected that polyelectrolytes with longer and more hydrophobic side chains would have stronger anti-adhesion properties (13).
Comparison of bacterial cell counts on control surfaces and surfaces coated with PEMs
Polyelectrolyte multilayers | Bacterial count (log/mm2) | Difference to control (A log no. bact./mm2) | Bacterial count (no./mm2) | Difference to control (%) | t-value | p-value |
---|---|---|---|---|---|---|
PAH/PSS 5 mmol/L | 3.784 | 0.079 | 6,316 | 3.577 | 0.0010* | |
PAH/PSS 50 mmol/L | 3.651 | 0.212 | 4,771 | 7.942 | 0.0000** | |
PVP-ethyl Br/PSS 5 mmol/L | 3.589 | 0.274 | 4,058 | 47.3 | 11.743 | 0.0000** |
PVP-ethyl Br/PSS 50 mmol/L | 3.466 | 0.397 | 3,075 | 60.0 | 16.637 | 0.0000** |
PVP-isobutyl Br/PSS 5 mmol/L | 3.726 | 0.137 | 5,504 | 29.8 | 6.163 | 0.0000** |
PVP-isobutyl Br/PSS 50 mmol/L | 3.641 | 0.222 | 4,750 | 38.4 | 6.138 | 0.0000** |
/ | / | / |
*p<0.05;**p<0.001
Microscopy of
However, the development of anti-microbial coatings should rely on the safe-by-design concept. This includes precautionary measures and tools to identify uncertainties and potential risks at the earliest feasible stage of development. Understanding antimicrobial toxicity of applied coatings or their production and surface application and durability is needed to assure safety. For example, the same antimicrobial coating if applied inappropriately may on different surfaces lead to a release of biocides due to incomplete chemical binding (26).
Science is facing new challenges in creating surfaces that would allow systematic management of the attachment of living cells. PEMs provide numerous coating opportunities that can be used to control bacterial attachment. Our study has demonstrated that the selection of proper PEM and proper layering and concentration can optimise anti-adhesive efficiency. In this respect, the best result (60 % reduction) was achieved with top-layer polycations with quaternary amine groups. This may encourage synthesising and evaluating new polyelectrolytes for better anti-adhesion coatings. At the same time, future research should assess potential toxic effects of PEMs.