Optotracing for selective fluorescence-based detection, visualization and quantification of live S. aureus in real-time

Abstract

Methods for bacterial detection are needed to advance the infection research and diagnostics. Based on conformation-sensitive fluorescent tracer molecules, optotracing was recently established for dynamic detection and visualization of structural amyloids and polysaccharides in the biofilm matrix of gram-negative bacteria. Here, we extend the use of optotracing for detection of gram-positive bacteria, focussing on the clinically relevant opportunistic human pathogen Staphylococcus aureus. We identify a donor-acceptor-donor-type optotracer, whose binding-induced fluorescence enables real-time detection, quantification, and visualization of S. aureus in monoculture and when mixed with gram-negative Salmonella Enteritidis. An algorithm-based automated high-throughput screen of 1920 S. aureus transposon mutants recognized the cell envelope as the binding target, which was corroborated by super-resolution microscopy of bacterial cells and spectroscopic analysis of purified cell wall components. The binding event was essentially governed by hydrophobic interactions, which permitted custom-designed tuning of the binding selectivity towards S. aureus versus Enterococcus faecalis by appropriate selection of buffer conditions. Collectively this work demonstrates optotracing as an enabling technology relevant for any field of basic and applied research, where visualization and detection of S. aureus is needed.

Structural Properties Dictating Selective Optotracer Detection of Staphylococcus aureus

“…Moreover, since the penta‐, hexa‐ and heptameric optotracers tested are equally charged, the hydrophobicity of individual molecules increased with increasing length. This supports our previous observation, showing the importance of hydrophobic interactions for binding to and detection of S. aureus [14] …”

“…This supports our previous observation, showing the importance of hydrophobic interactions for binding to and detection of S. aureus. [14] The charge of the optotracer influences detection of S. aureus…”

“…When found to differentiate between components of the extracellular matrix of bacterial biofilms [1,2] and polysaccharides in the glycobiology field, [11–13] the molecules were further investigated as optotracers. Recently, we demonstrated optotracer binding to cell wall components of Gram‐positive bacteria, e. g. peptidoglycan (PGN), thereby enabling selective detection of the pathogen Staphylococcus aureus ( S. aureus ) [14] . By introducing a donor‐acceptor‐donor (D‐A‐D)‐type electronic structure in the conjugated backbone of the optotracer, an on‐switch of fluorescence occurred, which enabled real‐time detection of bacterial cells in growing cultures [14] …”

“…Recently, we demonstrated optotracer binding to cell wall components of Gram-positive bacteria, e. g. peptidoglycan (PGN), thereby enabling selective detection of the pathogen Staphylococcus aureus (S. aureus). [14] By introducing a donoracceptor-donor (D-A-D)-type electronic structure in the conjugated backbone of the optotracer, an on-switch of fluorescence occurred, which enabled real-time detection of bacterial cells in growing cultures. [14] Here, we investigate the nature of the chemistry that promotes specific binding of optotracers to S. aureus.…”

Isolation and identification of Biofilm-producing E. coli from drinking water.

“…Hydrophobicity of biofilm producers was determined by the BATH method as described by Butina et al & Carniello et al 17,18 .…”