The synergistic effect of antimicrobial compounds is an important phenomenon that can increase the potency of treatment and might be useful against the formation of biofilms on surfaces. A strong inhibition of microbial viability on surfaces can potentially delay the development of biofilms on treated surfaces, thereby enhancing the performance of water-purification technologies and medical devices, for example, to prevent hospital-acquired infections. However, the synergistic effects of surface-immobilized antimicrobial peptides (AMPs) have not yet been reported. Here, we demonstrate the synergistic antimicrobial effects of the AMPs PGLa and magainin-2 on modified reverse-osmosis (RO) membranes. These AMPs are known to act synergistically in the free state, but their antimicrobial synergistic effects have not yet been reported in a surface-immobilized state. The AMPs were functionalized with alkyne linkers and covalently attached to RO membranes modified with azides, using a click chemistry reaction. The resulting RO membranes showed reduced contact angles, indicating increased wettability. X-ray photoelectron spectroscopy confirmed the presence of the two peptides on the membranes via changes in the amounts of carbon, oxygen, and sulfur, which led to an increased S/C ratio, probably because of the sulfur present in the methionine residue of the peptides. The synergistic activity was measured with the free peptides in solution and covalently bound on RO membrane surfaces by observing increased leakage of 5(6)-carboxyfluorescein from large unilamellar vesicles. The synergistic antimicrobial activity against Pseudomonas aeruginosa was observed using surface-activity assays, where the AMP-modified RO membranes showed an effective inhibition of P. aeruginosa biofilm growth, as compared with unmodified membranes. An enhanced activity of antimicrobials on surfaces might lead to potent antimicrobial surfaces, which could result in more fouling-resistant water-treatment membranes.
Bacterial biofilms that are formed on surfaces are highly detrimental to many areas of industry and medicine. Seawater desalination by reverse osmosis (RO) suffers from biofilm growth on the membranes (biofouling), which limits its widespread use because biofouling decreases water permeance and necessitates module cleaning and replacement, leading to increased economic and environmental costs. Antimicrobial peptides (AMPs) bound covalently to RO membranes inhibit biofilm growth and might delay membrane biofouling. Here we examined how various hydrophilic membrane coatings composed of zwitterionic, neutral, positively charged, and poly(ethylene glycol) (PEG)-grafted polymers affected the biocidal activity and the biofilm inhibition of a covalently bonded AMP on RO membranes. AMP magainin-2 was linked by the copper-catalyzed azide−alkyne cycloaddition reaction to a series of RO membranes that were grafted with different methacrylate polymers. Surface characterization by infrared spectroscopy, X-ray photoelectron spectroscopy, and water drop contact angle gave evidence of successful RO modifications, and zeta potential analysis reflected the increase in surface charge due to the linked, positively charged peptide. All AMP-modified membranes inhibited Pseudomonas aeruginosa growth compared to unmodified membranes, and the grafted methacrylic polymers did not significantly interfere with the peptide activity. On the other hand, membranes coated with zwitterionic and other acrylate polymers including AMP attachment inhibited biofilm growth more than either the AMP or the polymer coating alone. This enhancement led to ∼20% less biofilm biovolume on the membrane surfaces. The combination of antimicrobial coatings with polymer coatings known to resist fouling might aid future designs of surface coatings susceptible to biofilm growth.
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