The effects of different poly(ethylene glycol) (PEG) attachment strategies upon the adhesion of a Gramnegative bacteria (Pseudomonas sp.) was tested. PEG was covalently immobilized, at the lower critical solution temperature of PEG, to a layer of branched poly(ethylenimine) (PEI). PEI was both physically adsorbed to a stainless-steel (SS) substrate and covalently immobilized to a carboxylated poly(ethylene terephthalate) (PET-COOH) surface. On both substrates, the PEI and PEG grafting conditions were optimized so that the levels of surface coverage after each step were maximized and were the same on both substrates, as judged by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Also, ToF-SIMS imaging showed that both substrates were chemically uniform after each surface modification step. Thus, the two surfaces differ only in the mode of attachment of PEI to the substrate. In bacterial adhesion experiments, the optimal SS-PEG surface was not capable of reducing the number of adherent Pseudomonas sp. when compared to the controls. However, the PET-PEG surface reduced the level of adhesion by between 2 and 4 orders of magnitude for up to 5 h. ToF-SIMS analysis showed that both PEG surfaces adsorbed low but comparable levels of proteinaceous growth medium components (tryptic soy broth), as indicated by the addition of unique amino acid fragment ions in the spectra, most likely small peptides. Thus, bacterial adhesion was strongly dependent on the PEG immobilization strategy and not on the extent of peptide/protein adsorption. However, for the best PEG surfaces the residual bacterial adhesion is most likely from recognition of the small amount of adsorbed peptides. This highlights the necessity for preventing the adsorption of small biological species that can even penetrate PEG layers of high graft density, in the quest for the ultimate "nonfouling" surface.

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The fish probiotic bacterium Roseobacter strain 27-4 grows only as rosettes and produces its antibacterial compound under static growth conditions. It forms three-dimensional biofilms when precultured under static conditions. We quantified attachment of Roseobacter strain 27-4 using a direct real-time PCR method and demonstrated that the bacteria attached more efficiently to surfaces during static growth than under aerated conditions. Bacteria belonging to the marine Roseobacter clade may produce antibacterial compounds (4, 5, 6) and have been suggested for use as a probiotic treatment in aquaculture (12,19,23,24). Roseobacter strains were isolated from a turbot rearing facility, and very similar DNA types were repeatedly isolated from tank walls over a 1-year period (13). One of these strains, Roseobacter strain 27-4, inhibits fish pathogenic bacteria by a sulfur-containing antibacterial compound (5, 12) and improves the survival of turbot larvae infected with Vibrio anguillarum (19). The antibacterial compound is produced only under static growth conditions, where the bacterium forms a thick biofilm at the air-liquid interface. The biofilm produced by Roseobacter strain 27-4 appears to be correlated to a multicellular growth pattern in which 9 to 10 cells grow in a rosette shape. The rosette is unstable, and shaking will break up the structure; during growth under aerated conditions, the organism grows as single cells and no inhibitory activity is detected (5).It is not known if the probiotic Roseobacter strain 27-4 retains its antibacterial properties when it attaches to inert surfaces; however, marine bacteria such as Roseobacter gallaeciensis and Pseudoalteromonas tunicata can prevent other bacteria from forming a biofilm (20). We hypothesize that interaction between Roseobacter and fish pathogenic bacteria may take place on surfaces in biofilms and that it potentially can exert its effect as a probiotic bacterium by colonizing the turbot larva rearing facilities. Here we describe the development of a realtime PCR method that allows quantification of Roseobacter strain 27-4 on solid surfaces. We also describe how culture conditions may influence biofilm formation of Roseobacter strain 27-4.Effect of preculture growth conditions on pigmentation. In this study, as in previous studies (5), Roseobacter strain 27-4 grown in marine broth (MB) (Difco 279110) under static conditions grew as rosettes consisting of 9 to 10 cells, produced a brown pigment, and formed a biofilm at the air-liquid interface. However, if the statically grown preculture was vigorously shaken before inoculation or if an aerated preculture was used, almost no pigment and biofilm formation were produced (data not shown). Furthermore, when an aerated preculture was used, almost no rosette-forming cell clusters were observed even after several days of static growth.Roseobacter strain 27-4 biofilm formation on plastic surfaces. Statically grown overnight cultures of Roseobacter strain 27-4 grown in MB were diluted to an optical density at 600 nm...