Antimicrobial testing for surface‐immobilized agents with a surface‐separated live–dead staining method

Abstract

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 mm 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.

A review of immobilized antimicrobial agents and methods for testing

“…86,87 Our group has devised a conceptually simple live-dead staining technique that can determine whether the antimicrobial agent kills cells at the surface or at a distance. 88 The method uses a direct inoculation method with spacers to separate an iAMA surface from a control coverslip surface. The method generates three populations of bacteria that can be compared: (1) those at the control surface, (2) those at the test iAMA surface, and (3) those freely floating in the solution.…”

High-content analysis for drug delivery and nanoparticle applications

“…As a confirmation of the innocuous nature of pDMAEMA predicted by HCA, it did not induce any histological damage on isolated rat colonic mucosae and caused on only mild damage to ileal tissue at even higher concentrations of the agent, with exposure for a more realistic 120 min compared to 72 h with cell lines. Therefore, the data supported the potential use of pDMAEMA as a gene delivery agent [35] and as an antibacterial surface coating [36], with some confidence in the likelihood of a safe outcome from a full preclinical toxicology package. HCA has also been used to study the mechanisms of cytotoxicity for two of the most common cationic polymeric carriers, PEI and poly-L-lysine (PLL) [37].…”

Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria

“…To evaluate whether the eGFP/3 μM PI assay is suited for in situ monitoring of bacterial viability and growth on a bioactive model substrate, eGFP-expressing E. coli (AAEC191A pHis-GFP) were incubated on antimicrobial dimethyloctadecyl [3-(trimethoxysilyl)propyl]ammonium chloride (DMOAC) coated glass surfaces [23,27] (Figure 3b). Homogeneous DMOAC coating with a dry adlayer thickness of 2.2 nm was confirmed by variable-angle spectroscopic ellipsometry.…”

“…Bioactive surfaces were prepared according to published protocols [23]. Briefly, glass cover slides, that later resemble the bottom slide of the flow chamber, were exposed to air plasma for 15 seconds (Harrick Plasma, PDC-32G) followed by dipping into a 5% (v/v) aqueous DMOAC (Sigma-Aldrich) solution for 1 second, and drying at 105°C overnight.…”

“…Beyond calibrating the assay and monitoring E. coli surface colonization kinetics on bare glass substrates, we demonstrate that this assay is applicable to monitor the inactivation kinetics of E. coli in contact with antimicrobial surface coatings, using dimethyloctadecyl [3-(trimethoxysilyl)propyl]ammonium chloride (DMOAC) coated glass surfaces as model substrate. To kill bacteria, the quaternary ammonium chloride complexes of surface-bound DMOAC have to directly interact with the bacterial membrane [23]. We previously showed that bacterial fimbriae strongly influence the unspecific adhesion of E. coli to engineered surfaces [4].…”