Structural Properties Dictating Selective Optotracer Detection of Staphylococcus aureus

Abstract

Optotracers are conformation-sensitive fluorescent tracer molecules that detect peptide-and carbohydrate-based biopolymers. Their binding to bacterial cell walls allows selective detection and visualisation of Staphylococcus aureus (S. aureus).Here, we investigated the structural properties providing optimal detection of S. aureus. We quantified spectral shifts and fluorescence intensity in mixes of bacteria and optotracers, using automatic peak analysis, cross-correlation, and areaunder-curve analysis. We found that the length of the conjugated backbone and the number of charged groups, but not their distribution, are important factors for selective detection of S. aureus. The photophysical properties of optotracers were greatly improved by incorporating a donor-acceptor-donor (D-A-D)-type motif in the conjugated backbone. With significantly reduced background and binding-induced onswitch of fluorescence, these optotracers enabled real-time recordings of S. aureus growth. Collectively, this demonstrates that chemical structure and photophysics are key tunable characteristics in the development of optotracers for selective detection of bacterial species.

Optotracing for live selective fluorescence-based detection of Candida albicans biofilms

“…Optotracing utilizes a group of tracer molecules that emit no meaningful fluorescence in their unbound state. Binding to a target induces an on-like switching of the tracer alongside a unique fluorescence profile that can potentially identify each interaction (Choong et al, 2016a;Butina et al, 2020;Butina et al, 2022). With no reported toxicity against microbial and eukaryotic cells, optotracers enable real-time monitoring of culture growth and the transition from planktonic to a biofilm lifestyle (Choong et al, 2016a;Butina et al, 2022).…”

“…Binding to a target induces an on-like switching of the tracer alongside a unique fluorescence profile that can potentially identify each interaction (Choong et al, 2016a;Butina et al, 2020;Butina et al, 2022). With no reported toxicity against microbial and eukaryotic cells, optotracers enable real-time monitoring of culture growth and the transition from planktonic to a biofilm lifestyle (Choong et al, 2016a;Butina et al, 2022). Recently, we developed an optotracing method for the semi-high throughput detection and monitoring of Salmonella biofilms growth on air-solid interfaces (Choong et al, 2021).…”

Optotracers to study biofilms in host-relevant systems and non-conventional treatments usingStaphylococcus aureuswound biofilms as a case study

“…When added to biofilm growth conditions or assays, optotracers have been reported to detect both bacterial cells and biofilm extracellular polymeric substance (EPS) components, which enables the in situ visualization of biofilm formation, structure, and development [4][5][6]. Given this ability to visualize live biofilms in a non-disruptive manner, optotracers have been used to study biofilm formation and features in a range of pathogens, including Salmonella, Uropathogenic Escherichia coli (UPEC) and Staphylococcus aureus [1,2,4,6,7]. In addition to detection and visualization of biofilms, the use of optotracers has expanded to applications in biofilm antibiotic susceptibility assays and biofilm markers in clinical diagnostics [1,4,7].…”

“…Optotracers are fluorescent visualization molecules that can be used for the real-time detection, visualization, characterization, and quantification of biofilms [1][2][3]. When added to biofilm growth conditions or assays, optotracers have been reported to detect both bacterial cells and biofilm extracellular polymeric substance (EPS) components, which enables the in situ visualization of biofilm formation, structure, and development [4][5][6]. Given this ability to visualize live biofilms in a non-disruptive manner, optotracers have been used to study biofilm formation and features in a range of pathogens, including Salmonella, Uropathogenic Escherichia coli (UPEC) and Staphylococcus aureus [1,2,4,6,7].…”

An optotracer-based antibiotic susceptibility test specifically targeting the biofilm lifestyle of Salmonella

“…While such methods work well for end stage analysis, they are not capable of detecting biofilm formation in real-time. Answering to this need, we recently developed several non-disruptive methodologies based on optotracers, which are non-toxic, optically active fluorescent tracer molecules which when added to the nutrient broth or agar report bacterially produced polysaccharides and peptides in real-time [ [13] , [14] , [15] , [16] ]. By allowing real-time visualization and quantification of bacterial growth and biofilm development, including formation of the extracellular matrix (ECM), the optotracing methodologies have generated deeper understandings of the formation and composition of Salmonella [ 13 , 17 ], E. coli [ 14 , 17 ], and Burkholderia [ 18 ] biofilms.…”