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.Electronic supplementary materialThe online version of this article (doi:10.1186/1559-4106-8-22) contains supplementary material, which is available to authorized users.
Highlights
Relevant nosocomial infections are frequently caused by adherent bacteria and the subsequent biofilm formation within tissues or on biomaterial surfaces [1]
To confirm that the viable E. coli in the medium supplemented with 3 μM propidium iodide (PI) were able to replicate, we compared the turbidity increase of a 50% live / 50% isopropanol killed bacterial mixture to cultures containing 100% live and 100% isopropanol treated E. coli (Figure 2a)
Supplementing the medium with 30 μM PI significantly increased the PI fluorescence from the 100% live starting culture (Figure 2b), which was consistent with the impaired growth rate under those conditions (Figure 2a), indicating that 30 μM but not 3 μM PI is toxic to E. coli bacteria
Summary
Relevant nosocomial infections are frequently caused by adherent bacteria and the subsequent biofilm formation within tissues or on biomaterial surfaces [1]. When DNA intercalating dyes like SYTO® 9 and propidium iodide (PI) are used [18], the impact of the potentially toxic stains on bacterial physiology has to be considered to avoid false negative results [19] Since both stains target DNA, the competitive displacement of the SYTO® 9 (live stain) by the high affinity PI (dead stain) upon membrane breakdown can affect the staining reliability [20]. To eliminate the competitive displacement of the two DNA stains and the demand for prolonged incubation times caused by the passive diffusion of the SYTO® 9 live stain through the bacterial membrane, it was suggested to replace SYTO® 9 with green fluorescent protein (GFP) expressed by the bacteria as demonstrated previously for flow cytometry applications (Figure 1b) [21]. Flow cytometry has been used to measure the viability of GFP expressing bacteria adsorbed to polystyrene beads functionalized with antimicrobial coatings [22], it cannot be applied for continuous in situ bacterial viability monitoring on planar surfaces
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