title: Non-proteinaceous bacterial adhesins challenge the antifouling properties of polymer brush coatings.
abstract: Polymer brushes of poly(ethylene glycol) have long been considered the gold standard for antifouling surfaces that resist adsorption of biomolecules and attachment of microorganisms. However, despite displaying excellent resistance to protein adsorption, the polymer brush coatings cannot entirely avoid colonization by bacteria. Here we investigate and identify which non-proteinaceous bacterial adhesins challenge the antifouling properties of polymer brush coatings and how these challenges might be overcome. We quantified biofilm formation on a well-known polymer brush coating of poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) on titanium. The coating successfully resisted colonization by Staphylococcus aureus and Pseudomonas aeruginosa, but not Staphylococcus epidermidis. This colonization pattern was also reflected on the adhesion forces measured on single bacterial cells. The biofilm produced from S. epidermidis on PLL-g-PEG were found to be rich in polysaccharides and extracellular DNA, and quantification of DNA, polysaccharides and proteins on PLL-g-PEG surfaces revealed that although the coating almost fully resisted protein adsorption, polysaccharides could adsorb, and exposure to DNA led to desorption of the polymer from the titanium surface. We hypothesized that this problem could be overcome by increasing the polymer brush density to better resist the penetration of DNA and polysaccharides into the polymer layer. Indeed, high density PLL-g-PEG brushes prepared by the recently discovered temperature-induced polyelectrolyte (TIP) grafting method resisted the interaction with DNA and polysaccharides, and therefore also the colonization by S. epidermidis. The TIP grafting is a simple improvement of PLL-g-PEG brush formation, and our results suggest that it provides an important advancement to the bacterial resistance by polymer brush coatings.
  
  
  CONCLUSIONS
  The antifouling properties of poly(ethylene glycol) brush coatings against protein adsorption are well documented, but it is not well understood why these coatings do not perform as well against bacterial colonization when tested against a wide range of species and over periods of days. Here we investigated bacterial colonization on poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) grafted on Ti, and revealed that bacteria relying mostly on polysaccharides and extracellular DNA for adhesion and biofilm formation could successfully colonize PLL-g-PEG coated surfaces. The coatings could not resist adsorption of polysaccharides, and DNA could even desorb the coatings from the Ti surface. Fortunately, the shortcomings of conventional PLL-g-PEG could be overcome by increasing the graft density, using the recently discovered and very simple grafting method, 'temperature-induced polyelectrolyte (TIP) grafting'. Our study highlights that it is of utmost importance to develop coatings which resist adsoprtion of non-proteinaceous bacterial adhesins such as polysaccharides and DNA, and we demonstrated that TIP grafted high density PLL-g-PEG coatings are promising materials to achieve diverse bacterial resistance.
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Faculty of Science and Technology, Aarhus University, Aarhus 8000, Denmark.
authors.forename: Guanghong
authors.initials: G
authors.lastname: Zeng
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Faculty of Science and Technology, Aarhus University, Aarhus 8000, Denmark. Electronic address: ryo@inano.au.dk.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Ogaki
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Faculty of Science and Technology, Aarhus University, Aarhus 8000, Denmark; Department of Bioscience, Faculty of Science and Technology, Aarhus University, Aarhus 8000, Denmark. Electronic address: rikke.meyer@inano.au.dk.
authors.forename: Rikke L
authors.initials: RL
authors.lastname: Meyer
mesh_terms: D018829:Adhesins, Bacterial / Q000378:metabolism*; D001422:Bacterial Adhesion / Q000502:physiology; D018441:Biofilms / Q000254:growth & development*; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D013211:Staphylococcus aureus / Q000502:physiology*; D013212:Staphylococcus epidermidis / Q000502:physiology*; D014025:Titanium / Q000737:chemistry
chemical_list: D018829:Adhesins, Bacterial; D020099:Coated Materials, Biocompatible; D011135:Polysaccharides, Bacterial; C055239:bacterial adhesins, polysaccharide; D011107:Polylysine; D011092:Polyethylene Glycols; D014025:Titanium; C000595209:polyethylene glycol 1000
keywords: AFM; Bacterial adhesion; Biofilm; PLL-g-PEG; Polymer brush