Review of immobilized antimicrobial agents and methods for testing.

Antimicrobial surfaces for food and medical applications have historically involved antimicrobial coatings that elute biocides for effective kill in solution or at surfaces. However, recent efforts have focused on immobilized antimicrobial agents in order to avoid toxicity and the compatibility and reservoir limitations common to elutable agents. This review critically examines the assorted antimicrobial agents reported to have been immobilized, with an emphasis on the interpretation of antimicrobial testing as it pertains to discriminating between eluting and immobilized agents. Immobilization techniques and modes of antimicrobial action are also discussed.

Antimicrobial testing for surface-immobilized agents with a surface-separated live-dead staining method.

Modification of a traditional live-dead staining technique based on fluorescence microscopy has yielded an improved method capable of differentiating surface-immobilized antimicrobial agents from those agents acting via solution diffusion processes. By utilizing an inoculation chamber comprised of 50 µm polystyrene spheres as spacers between test substrate and coverslip control surfaces, three distinct bacterial cell populations can be probed by fluorescence microscopy for antimicrobial activity: (1) cells adhered to the coverslip, (2) cells adhered to the substrate, and (3) mobile cells in solution. Truly immobilized antimicrobial agents were found efficacious only at the substrate surface, while elutable agents were effective against all three populations. Glass surfaces derivatized with either quaternized poly dimethylaminoethylmethacrylate (pDMAEMA) or 3-(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (Si-QAC) were compared with bare glass control surfaces after contact and 4 h incubation with Staphylococcus aureus. pDMAEMA surfaces were both antimicrobial and immobilized, whereas the Si-QAC surfaces were only observed to be antimicrobial via active diffusion. In contrast to conventional thinking, Si-QAC surfaces showed no kill after removing all Si-QAC elutables via rinsing procedures. The semi-quantitative surface-separated live-dead staining (SSLDS) technique provides mechanistic insight and represents a significant improvement relative to current microbiological test methods for evaluating immobilized, antimicrobial agents.