Poly(ethylene glycol)-Based Coatings Combining Low-Biofouling and Quorum-Sensing Inhibiting Properties to Reduce Bacterial Colonization

Infections resulting from the formation of biofilms on medical devices remain a significant clinical problem. There is growing consensus that coatings displaying multiple defense mechanisms, such as low biofouling combined with surface active antimicrobial agents, is required. Quorum sensing (QS) is a bacterial mechanism used to coordinate their collective behavior. QS can also be exploited for antimicrobial purposes, to minimize colonization and biofilm formation by hindering bacterial communication. We have investigated a poly­(ethylene glycol) (PEG) based multifunctional coating that allows the covalent incorporation of the synthetic QS inhibitor 5-methylene-1-(prop-2-enoyl)-4-(2-fluorophenyl)-dihydropyrrol-2-one (DHP) with a surface providing reduced cell attachment and bacterial adhesion. The simple coating, which can be applied using either a one- or two-step procedure, provides the first example for a multifunctional surface offering a combination of a quorum sensing inhibitor with a low biofouling background. X-ray photoelectron spectroscopy (XPS) was utilized to confirm the coating formation and the incorporation of DHP. L929 mouse fibroblast cell attachment and cytotoxicity studies demonstrated the low biofouling and biocompatible properties of the coatings. Bacterial colonization assays using <i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i> demonstrated the ability of these combination coatings to reduce the formation of biofilms. Importantly, the results demonstrate that the DHP remained active after covalent incorporation into the coating

Expression of cytokeratins (CK) and GAPDH in HMGEC.

<p>(A) Representative western blots show specific bands for GAPDH, CK1, CK5, CK6 and CK14. (B) Intensities (normalized to GAPDH) are shown as mean ± SEM and statistical significance vs. 21d serum treatment is indicated by asterisks (n = 6, one-way ANOVA; * <i>p <</i> 0.05).</p

Sudan III staining for lipid detection in HMGEC.

<p>Cells were either cultured in serum-free medium until they reached 70–90% confluence (A, B) or cultured in serum-containing medium for 1 (C), 7 (D) or 14 (E) days. The graph in (F) shows quantification of Sudan III stained areas normalized to the cell count per image. Serum-free treated cells showed no lipid accumulation. Cells in serum-containing media accumulated lipids in the cytoplasm after one day treatment. Lipid accumulation decreased over time. The red stain in the pictures indicates lipid droplets.</p

Additional file 1 of Assessment of genotypes, endosymbionts and clinical characteristics of Acanthamoeba recovered from ocular infection

Additional file 1: Table S1: Reference Acanthamoeba strains used in this study for phylogenetic analysis. Table 2: Genotype and species identification of Acanthamoeba isolates recovered from AK patients. Map 1: Map showing the states of the AK patients from different states of India with sample codes and identified genotypes of Acanthamoeba from AK patients. The map was created using ArcGIS (Esri GIS, California, USA). Fig. S1. Monthly distribution of AK cases during study period. Fig. S2. Sequence alignment of Acanthamoeba 18S rDNA DF3 region using ClustalW. Table S3. Overall clinical presentation of the keratitis patients infected with Acanthamoeba spp. Fig S3. Phylogenetic tree inferred from the 18S (ITS1) rDNA sequence of fungi; tree was created using the neighbour-joining approach with the Kimura 2-parameter based on 1000 replicates bootstrap values. Fig. S4. Agarose (1%) gel image of PCR amplicons of 13 Acanthamoeba isolates (18S rRNA), PCR assay was performed using Acanthamoeba genus specific primer pair JDPFw and JDPRv which yielded ~450bp amplicons. Fig. S5: Agarose (1%) gel image of PCR amplicons of 13 Acanthamoeba isolates targeting intracellular bacteria 16S rRNA, primer pair 515Fw and 806Rv (V4, 16S rRNA) was used which yielded ~293bp amplicons

Ultra-structural analysis of HMGEC.

<p>Cells were either cultured in serum-free medium until they reached 90% confluence (A) or cultured in serum-containing medium for 1 (B, higher magnification in F), 3 (C), 7 (D) or 14 (E) days. Cytokeratin filaments (CF) elongated and desmosomes (black arrow heads) increased in serum-treated cells over time, but desmosomes were not visible in cells grown in serum-free media. Lipid droplets (arrows) can be seen in serum-treated cells. N = nuclei, ES = extracellular space.</p

Comparison of lipids reported in the current study to those reported for HMGECs [4] and epithelial cells [37] in previous reports.

<p>phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM), diacylglycerol (DAG), ceramide (Cer), cholesterol ester (CE), free cholesterol (Chol), and wax ester (WE)</p><p>Comparison of lipids reported in the current study to those reported for HMGECs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128096#pone.0128096.ref004" target="_blank">4</a>] and epithelial cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128096#pone.0128096.ref037" target="_blank">37</a>] in previous reports.</p

A. Sudan III staining to visualize lipid accumulation of HMGEC after 1 day or 7 days stimulation with addition of 10% FCS (C, D), high glucose (E, F), lipid cocktail (G, H), 100μM EPA (I, J) in10% serum-containing medium (A, B) or sebomed medium (K, L).

<p>Fig 6M shows quantification of Sudan III stained areas normalized to the cell count per image. In general, lipid accumulations were more prominent after 1 day compared to 7 days cultivation in serum-containing medium. Highest levels of lipids were visible after 1 day treatment with 100μM EPA (I). Red stain indicates lipid droplets.</p

Ultra-structural analysis of HMGEC after 1 day stimulation with serum-containing medium supplemented with 100 μM EPA (A) and 20% FCS (B).

<p>Cytokeratin filaments (CF), desmosomes (black arrow heads) and lipid droplets (arrows) are visible in 20% FCS treated cells. EPA stimulated cells show numerous lysosomes (white arrow heads).</p