Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinan
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ORIGINAL ARTICLE
Open Access
Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria Ima Avalos Vizcarra1, Philippe Emge1, Philipp Miermeister2, Mamta Chabria1, Rupert Konradi3, Viola Vogel1 and Jens Möller1*
Abstract Bacterial adhesion and biofilm growth can cause severe biomaterial-related infections and failure of medical implants. To assess the antifouling properties of engineered coatings, advanced approaches are needed for in situ monitoring of bacterial viability and growth kinetics as the bacteria colonize a surface. Here, we present an optimized protocol for optical real-time quantification of bacterial viability. To stain living bacteria, we replaced the commonly used fluorescent dye SYTO® 9 with endogenously expressed eGFP, as SYTO® 9 inhibited bacterial growth. With the addition of nontoxic concentrations of propidium iodide (PI) to the culture medium, the fraction of live and dead bacteria could be continuously monitored by fluorescence microscopy as demonstrated here using GFP expressing Escherichia coli as model organism. The viability of bacteria was thereby monitored on untreated and bioactive dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAC)-coated glass substrates over several hours. Pre-adsorption of the antimicrobial surfaces with serum proteins, which mimics typical protein adsorption to biomaterial surfaces upon contact with host body fluids, completely blocked the antimicrobial activity of the DMOAC surfaces as we observed the recovery of bacterial growth. Hence, this optimized eGFP/PI viability assay provides a protocol for unperturbed in situ monitoring of bacterial viability and colonization on engineered biomaterial surfaces with single-bacteria sensitivity under physiologically relevant conditions. Keywords: Antimicrobial surfaces; Optical viability monitoring; Green fluorescent protein (GFP); SYTO® 9; Propidium iodide (PI)
Background Clinically relevant nosocomial infections are frequently caused by adherent bacteria and the subsequent biofilm formation within tissues or on biomaterial surfaces [1]. Surface biofouling commonly starts with the adhesion of individual bacteria that subsequently grow into mature biofilms. To prevent bacterial adhesion and growth already during the pre-biofilm phase, two main surface engineering strategies have been developed so far: non-fouling “stealth” surface coatings that inhibit adhesion of proteins and bacteria [2-4] and bioactive materials, which upon bacterial contact or release of the active molecules interfere with bacterial viability [5-10]. To compare the antimicrobial * Correspondence: [email protected] 1 Department of Health Sciences and Technology, Laboratory of Applied Mechanobiology, ETH Zurich, CH-8093 Zurich, Switzerland Full list of author information is available at the end of the article
properties of surface coatings and to study the kinetics of bacterial surface colonization, assays are needed that allow for in situ monitoring
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