Coating of silicone with mannoside-PAMAM dendrimers to enhance formation of non-pathogenic Escherichia coli biofilms against colonization of uropathogens.

Bacterial interference using non-pathogenic Escherichia coli 83972 is a novel strategy for preventing catheter-associated urinary tract infection (CAUTI). Crucial to the success of this strategy is to establish a high coverage and stable biofilm of the non-pathogenic bacteria on the catheter surface. However, this non-pathogenic strain is sluggish to form biofilms on silicone as the most widely used material for urinary catheters. We have addressed this issue by modifying the silicone catheter surfaces with mannosides that promote the biofilm formation, but the stability of the non-pathogenic biofilms challenged by uropathogens over long-term remains a concern. Herein, we report our study on the stability of the non-pathogenic biofilms grown on propynylphenyl mannoside-modified silicone. The result shows that 94% non-pathogenic bacteria were retained on the modified silicone under >0.5 Pa shear stress. After being challenged by three multidrug-resistant uropathogenic isolates in artificial urine for 11 days, large amounts (>4 × 106 CFU cm-2) of the non-pathogenic bacteria remained on the surfaces. These non-pathogenic biofilms reduced the colonization of the uropathogens by >3.2-log. STATEMENT OF SIGNIFICANCE: In bacterial interference, the non-pathogenic Escherichia coli strains are sluggish to form biofilms on the catheter surfaces, due to rapid removal by urine flow. We have demonstrated a solution to this bottleneck by pre-functionalization of mannosides on the silicone surfaces to promote E. coli biofilm formation. A pre-conjugated high affinity propynylphenyl mannoside ligand tethered to the nanometric amino-terminated poly(amido amine) (PAMAM) dendrimer is used for binding to a major E. coli adhesin FimH. It greatly improves the efficiency for the catheter modification, the non-pathogenic biofilm coverage, as well as the (long-term) stability for prevention of uropathogen infections.

Probiotic E. coli Nissle 1917 biofilms on silicone substrates for bacterial interference against pathogen colonization.

Bacterial interference is an alternative strategy to fight against device-associated bacterial infections. Pursuing this strategy, a non-pathogenic bacterial biofilm is used as a live, protective barrier to fence off pathogen colonization. In this work, biofilms formed by probiotic Escherichia coli strain Nissle 1917 (EcN) are investigated for their potential for long-term bacterial interference against infections associated with silicone-based urinary catheters and indwelling catheters used in the digestive system, such as feeding tubes and voice prostheses. We have shown that EcN can form stable biofilms on silicone substrates, particularly those modified with a biphenyl mannoside derivative. These biofilms greatly reduced the colonization by pathogenic Enterococcus faecalis in Lysogeny broth (LB) for 11days. STATEMENT OF SIGNIFICANCE: Bacterial interference is an alternative strategy to fight against device-associated bacterial infections. Pursuing this strategy, we use non-pathogenic bacteria to form a biofilm that serves as a live, protective barrier against pathogen colonization. Herein, we report the first use of preformed probiotic E. coli Nissle 1917 biofilms on the mannoside-presenting silicone substrates to prevent pathogen colonization. The biofilms serve as a live, protective barrier to fence off the pathogens, whereas current antimicrobial/antifouling coatings are subjected to gradual coverage by the biomass from the rapidly growing pathogens in a high-nutrient environment. It should be noted that E. coli Nissle 1917 is commercially available and has been used in many clinical trials. We also demonstrated that this probiotic strain performed significantly better than the non-commercial, genetically modified E. coli strain that we previously reported.

Surfaces presenting α-phenyl mannoside derivatives enable formation of stable, high coverage, non-pathogenic Escherichia coli biofilms against pathogen colonization.

Prevention of pathogenic colonization on medical devices over a long period of time remains a great challenge, especially in a high-nutrient environment that accelerates the production of biomass leading to biofouling of the device. Since biofouling and the subsequent pathogen colonization is eventually inevitable, a new strategy using non-pathogenic bacteria as living guards against pathogenic colonization on medical devices has attracted increasing interest. Crucial to the success of this strategy is to pre-establish a high coverage and stable biofilm of benign bacteria on the surface. Silicone elastomers are one of the most widely used materials in biomedical devices. In this work, we modified silicone surfaces to promote formation of high coverage and stable biofilms by a non-pathogenic Escherichia coli strain 83972 with type 1 fimbriae (fim+) to interfere with the colonization of an aggressive biofilm-forming, uropathogenic Enterococcus faecalis. Although it is well known that mannoside surfaces promote the initial adherence of fim+ E. coli through binding to the FimH receptor at the tip of the type 1 fimbriae, it is not clear whether the fast initial adherence could lead to a high coverage and stable protective biofilm. To explore the role of mannoside ligands, we synthesized a series of alkyl and aryl mannosides varied in the structure and immobilized them on silicone surfaces pre-coated with a poly(amidoamine) (PAMAM) dendrimer. We found that stable and densely packed benign E. coli biofilms were formed on the surfaces presenting biphenyl mannoside with the highest initial adherence of fim+ E. coli. These non-pathogenic biofilms prevented the colonization of E. faecalis for 11 days at a high concentration (10(8) CFU mL(-1), 100,000 times above the diagnostic threshold for urinary tract infection) in the nutrient-rich Lysogeny Broth (LB) media. The result shows a correlation among the initial adherence of fim+ E. coli 83972, the coverage and long-term stability of the resulting biofilms, as well as their efficiency for preventing the pathogen colonization.

Surfaces Presenting α-Phenyl Mannoside Derivatives Enable Formation of Stable, High Coverage, Non-pathogenic Escherichia coli Biofilms against Pathogen Colonization.

Prevention of pathogenic colonization on medical devices over a long period of time remains a great challenge, especially in a high-nutrient environment that accelerates production of biomass leading to biofouling of the device. Since biofouling and the subsequent pathogen colonization is eventually inevitable, a new strategy using non-pathogenic bacteria as living guards against pathogenic colonization on medical devices has attracted increasing interest. Crucial to the success of this strategy is to pre-establish a high coverage and stable biofilm of benign bacteria on the surface. Silicone elastomers are one of the most widely used materials in biomedical devices. In this work, we modified silicone surfaces to promote formation of high coverage and stable biofilms by a non-pathogenic Escherichia coli strain 83972 with type 1 fimbriae (fim+) to interfere the colonization of an aggressive biofilm-forming, uropathogenic Enterococcus faecalis. Although it is well known that mannoside surfaces promote the initial adherence of fim+ E. coli through binding to the FimH receptor at the tip of the type 1 fimbriae, it is not clear whether the fast initial adherence could lead to a high coverage and stable protective biofilm. To explore the role of mannoside ligands, we synthesized a series of alkyl and aryl mannosides varied in structure and immobilized them on silicone surfaces pre-coated with poly(amidoamine) (PAMAM) dendrimer. We found that stable and densely packed benign E. coli biofilms were formed on the surfaces presenting biphenyl mannoside with the highest initial adherence of fim+ E. coli. These non-pathogenic biofilms prevented the colonization of E. faecalis for 11 days at a high concentration (108 CFU mL-1, 100,000 times above the diagnostic threshold for urinary tract infection) in the nutrient-rich Lysogeny Broth (LB) media. The result shows a correlation among the initial adherence of fim+ E. coli 83972, the coverage and long-term stability of the resultant biofilms, as well as their efficiency for preventing the pathogen colonization.

Poly(ethylene imine)s as antimicrobial agents with selective activity.

We report the structure-activity relationship in the antimicrobial activity of linear and branched poly(ethylene imine)s (L- and B-PEIs) with a range of molecular weights (MWs) (500-12,000). Both L- and B-PEIs displayed enhanced activity against Staphylococcus aureus over Escherichia coli. Both B- and L-PEIs did not cause any significant permeabilization of E. coli cytoplasmic membrane. L-PEIs induced depolarization of S. aureus membrane although B-PEIs did not. The low MW B-PEIs caused little or no hemolysis while L-PEIs are hemolytic. The low MW B-PEIs are less cytotoxic to human HEp-2 cells than other PEIs. However, they induced significant cell viability reduction after 24 h incubation. The results presented here highlight the interplay between polymer size and structure on activity.

Nanoscale surface modification favors benign biofilm formation and impedes adherence by pathogens.

We have found in vitro that a biofilm of benign Escherichia coli 83972 interferes with urinary catheter colonization by pathogens, and in human studies E. coli 83972-coated urinary catheters are associated with lower rates of catheter-associated urinary tract infections. We hypothesized that modifying surfaces to present mannose ligands for the type 1 fimbriae of E. coli would promote formation of dense E. coli 83972 biofilms, thereby interfering with surface colonization by Enterococcus faecalis, a common uropathogen. We covalently immobilized mannose on silicon substrates by attaching amino-terminated mannose derivative to carboxylic acid-terminated monolayers via amidation. Fluorescence microscopy showed that E. coli 83972 adherence to mannose-modified surfaces increased 4.4-fold compared to unmodified silicon surfaces. Pre-exposing mannose-modified surfaces to E. coli 83972 established a protective biofilm that reduced E. faecalis adherence by 83-fold. Mannose-fimbrial interactions were essential for the improved E. coli 83927 adherence and interference effects. From the Clinical Editor: Recurrent urinary tract infections remain major adverse events associated with catheter use. The authors report that modifying catheter surface to present mannose ligands for the type 1 fimbriae of benign Escherichia coli 83972 promotes formation of dense E. coli biofilms, which 100-fold reduces urinary catheter colonization of uropathogens. Future application of this technology is expected to result in substantial UTI risk reduction in catheter users.

Biofunctionalization of silicone polymers using poly(amidoamine) dendrimers and a mannose derivative for prolonged interference against pathogen colonization.

Despite numerous preventive strategies on bacterial adhesion, pathogenic biofilm formation remained the major cause of medical device-related infections. Bacterial interference is a promising strategy that uses pre-established biofilms of benign bacteria to serve as live, protective coating against pathogen colonization. However, the application of this strategy to silicone urinary catheters was hampered by low adherence of benign bacteria onto silicone materials. In this work, we present a general method for biofunctionalization of silicone (PDMS) as one of the most widely used materials for biomedical devices. We used mild CO(2) plasma to activate PDMS surface followed by simple attachment of generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers to generate an amino-terminated surface that were maintained even after storage in PBS buffer for 36 days. We then covalently attach a carboxy-terminated mannose derivative to the modified PDMS to promote the adherence of benign Escherichia coli 83972 expressing mannose-binding type 1 fimbriae. We demonstrated that dense, stable biofilms of E. coli 83972 could be established within 48 h on the mannose-coated PDMS. Significantly, this benign biofilm reduced the adherence of the uropathogenic Enterococcus faecalis by 104-fold after 72 h, while the benign bacteria on the unmodified substrate by only 5.5-fold.

"Click" immobilization on alkylated silicon substrates: model for the study of surface bound antimicrobial peptides.

We describe an effective approach for the covalent immobilization of antimicrobial peptides (AMPs) to bioinert substrates via Cu(I) -catalyzed azide-alkyne cycloaddition (CuAAC). The bioinert substrates were prepared by surface hydrosilylation of oligo(ethylene glycol) (OEG) terminated alkenes on hydrogen-terminated silicon surfaces. To render the OEG monolayers "clickable", mixed monolayers were prepared using OEG-alkenes with and without a terminal alkyne protected by a trimethylgermanyl (TMG) group. The mixed monolayers were characterized by X-ray photoelectron spectroscopy (XPS), elliposometry and contact angle measurement. The TMG protecting group can be readily removed to yield a free terminal alkyne by catalytic amounts of Cu(I) in an aqueous media. This step can then be combined with the subsequent CuAAC reaction. Thus, the immobilization of an azide modified AMP (N3-IG-25) was achieved in a one-pot deprotection/coupling reaction. Varying the ratio of the two alkenes in the deposition mixture allowed for control over the density of the alkynyl groups in the mixed monolayer, and subsequently the coverage of the AMPs on the monolayer. These samples allowed for study of the dependence of antimicrobial activities on the AMP density. The results show that a relative low coverage of AMPs (∼1.6×10(13) molecule per cm(2)) is sufficient to significantly suppress the viability of Pseudomonas aeruginosa, while the surface presenting the highest density of AMPs (∼2.8×10(13) molecule per cm(2)) is still cyto-compatible. The remarkable antibacterial activity is attributed to the long and flexible linker and the site-specific "click" immobilization, which may facilitate the covalently attached peptides to interact with and disrupt the bacterial membranes.

Antibacterial activity and cytotoxicity of PEGylated poly(amidoamine) dendrimers.

We have investigated the antibacterial activity and cytotoxicity of a series of amino-terminated poly(amidoamine) (PAMAM) dendrimers modified with poly(ethylene glycol) (PEG) groups. The antibacterial activity of the PAMAM dendrimers and their derivatives against the common ocular pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, was evaluated by their minimum inhibitory concentrations (MICs). For the unmodified third and fifth generation (G3 and G5) amino-terminated dendrimers, the MICs against both P. aeruginosa and S. aureus were in the range of 6.3-12.5 microg mL(-1), comparable to that of the antimicrobial peptide LL-37 (1.3-12.5 microg mL(-1)) and within the wide range of 0.047-128 microg mL(-1) for the fluoroquinolone antibiotics. PEGylation of the dendrimers decreased their antibacterial activities, especially for the Gram-positive bacteria (S. aureus). The reduction in potency is likely due to the decrease in the number of protonated amino groups and shielding of the positive charges by the PEG chains, thus decreasing the electrostatic interactions of the dendrimers with the negatively-charged bacterial surface. Interestingly, localization of a greater number of amino groups on G5 vs. G3 dendrimers did not improve the potency. Significantly, even a low degree of PEGylation, e.g. 6% with EG(11) on G3 dendrimer, greatly reduced the cytotoxicity towards human corneal epithelial cells while maintaining a high potency against P. aeruginosa. The cytotoxicity of the PEGylated dendrimers to host cells is much lower than that reported for antimicrobial peptides. Furthermore, the MICs of these dendrimers against P. aeruginosa are more than two orders of magnitude lower than other antimicrobial polymers reported to date. These results motivate further exploration of the potential of cationic dendrimers as a new class of antimicrobial agents that may be less likely to induce bacterial resistance than standard antibiotics.