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

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.

Identification of Antimicrobial Peptides and Immobilization Strategy Suitable for a Covalent Surface Coating with Biocompatible Properties

“…Furthermore, even a lytic event can be suspected, which could lead to an alteration of the antimicrobial surface by the adsorption of biomolecules especially phosphoglycerides from bacterial membranes or proteins, which may be released upon cell lysis. This may influence the performance of the antimicrobial surface coating by shielding the antimicrobial peptides from living bacteria, which would result in the loss of activity . However, an adsorption of bacteria could not be proven by surface analyses using fluorescence microscopy.…”

Cationic Lignin as an Efficient and Biorenewable Antimicrobial Material

“…For CLSM imaging, the cells were stained with SYTO9 and PI dyes after treatment with QAL. Both of these are DNA-intercalating dyes but STYO9 (green dye) can diffuse passively into living bacteria, while PI (red dye) cannot pass an intact bacterial membrane and only enters permeabilized dead bacteria . Therefore, if the cells are predominantly green, it suggests that cells are alive, whereas if the cells are predominantly red, this likely suggests that the cell membrane is damaged allowing the permeation of the red dye.…”

“…Both of these are DNAintercalating dyes but STYO9 (green dye) can diffuse passively into living bacteria, while PI (red dye) cannot pass an intact bacterial membrane and only enters permeabilized dead bacteria. 110 Therefore, if the cells are predominantly green, it suggests that cells are alive, whereas if the cells are predominantly red, this likely suggests that the cell membrane is damaged allowing the permeation of the red dye. On the other hand, if the cells are orange, a partial damage of the cells allowing penetration of a few red dyes alongside green dyes can be inferred.…”

A microfluidic platform for in situ investigation of biofilm formation and its treatment under controlled conditions

“…To demonstrate the versatility of the developed platform, E. coli biofilms were treated with colistin, an effective antibiotic against Gram-negative bacteria, and the killing action was followed in real-time by using a method described by Avalos Vizcarra et al [27]. Briefly, propidium iodide (PI) was added to the antibiotic solution at a non-cytotoxic concentration.…”