Cationic antimicrobial peptides (cAMPs) kill bacteria in solution by membrane lysis; however, translating cAMPs into a covalently attached antibacterial coating is challenging since it remains unclear how the specifics of the conjugation impact the antifouling efficacy. Furthermore, studies have typically assessed cAMP coatings with a high and homogeneous surface coverage, although this may be difficult to implement in practice of the materials commonly used in medicine. Herein, we investigate the antifouling efficacy of fractional surface coatings made from poly(ethylene glycol) (PEG)-tethered cAMPs presented on gold nanoparticles (AuNPs) deposited onto surfaces. For all tested cAMPs, the antifouling efficacy increases exponentially with the 2D surface coverage of the coating. However, although the cAMPs have a similar primary sequence and display similar potency against Staphylococcus epidermidis in solution, the cyclic peptide is much more potent after tethering to the AuNPs than the linear counterparts. The attachment of the cyclic cAMPs also led to an unexpected shrinkage of the modified PEG-brush by more than 50%, indicating a restricted mobility of the tethering PEG chains. The shrinkage increased the closeness of the peptide on the AuNP and may thus enable cooperative actions of the grafted cAMPs such as the formation of nanosized peptide clusters that were previously found to enhance cAMP potency in solution. These findings pave the way for antibacterial coatings that cover only a subfraction of a material while remaining active in a clinical setting. 

Antimicrobial peptides (AMPs) can kill bacteria by destabilizing their membranes, yet translating these molecules’ properties into a covalently attached antibacterial coating is challenging. Rational design efforts are obstructed by the fact that standard microbiology methods are ill-designed for the evaluation of coatings, disclosing few details about why grafted AMPs function or do not function. It is particularly difficult to distinguish the influence of the AMP’s molecular structure from other factors controlling the total exposure, including which type of bonds are formed between bacteria and the coating and how persistent these contacts are. Here, we combine label-free live-cell microscopy, microfluidics, and automated image analysis to study the response of surface-bound Escherichia coli challenged by the same small AMP either in solution or grafted to the surface through click chemistry. Initially after binding, the grafted AMPs inhibited bacterial growth more efficiently than did AMPs in solution. Yet, after 1 h, E. coli on the coated surfaces increased their expression of type-1 fimbriae, leading to a change in their binding mode, which diminished the coating’s impact. The wealth of information obtained from continuously monitoring the growth, shape, and movements of single bacterial cells allowed us to elucidate and quantify the different factors determining the antibacterial efficacy of the grafted AMPs. We expect this approach to aid the design of elaborate antibacterial material coatings working by specific and selective actions, not limited to contact-killing. This technology is needed to support health care and food production in the postantibiotic era.