A Phage Receptor-Binding Protein as a Promising Tool for the Detection of Escherichia coli in Human Specimens

Abstract

Escherichia coli is a problematic pathogen that causes life-threatening diseases, being a frequent causative agent of several nosocomial infections such as urinary tract and bloodstream infections. Proper and rapid bacterial identification is critical for allowing prompt and targeted antimicrobial therapy. (Bacterio)phage receptor-binding proteins (RBPs) display high specificity for bacterial surface epitopes and, therefore, are particularly attractive as biorecognition elements, potentially conferring high sensitivity and specificity in bacterial detection. In this study, we elucidated, for the first time, the potential of a recombinant RBP (Gp17) to recognize E. coli at different viability states, such as viable but not culturable cells, which are not detected by conventional techniques. Moreover, by using a diagnostic method in which we combined magnetic and spectrofluorimetric approaches, we demonstrated the ability of Gp17 to specifically detect E. coli in various human specimens (e.g., whole blood, feces, urine, and saliva) in about 1.5 h, without requiring complex sample processing.

A perfect fit: Bacteriophage receptor-binding proteins for diagnostic and therapeutic applications

“…The assay could detect VBNC E. coli cells in different clinical matrices (e.g. blood and urine), providing a clear advantage over conventional culture-based methods [48]. As a final example, a Salmonella enterica detection system using the LPS-binding Det7 TSP (Det7T) was developed.…”

A microfluidic platform combined with bacteriophage receptor binding proteins for multiplex detection of Escherichia coli and Pseudomonas aeruginosa in blood

“…E. coli BL21 was purchased from Invitrogen. P. aeruginosa HB10 was utilized as the target bacteria for mAmetrine-Gp54 and as the negative control for mCherry-Gp17 [66]. E. coli HB106 was used as the target bacteria for mCherry-Gp17 protein and as the negative control for experiments using mAmetrine-Gp54.…”

“…The E. coli RBP (Gp17) used in this study was previously identified and synthesized fused with mCherry (mCherry-Gp17) [66]. The proteins were expressed and purified according to Costa et al [66].…”

“…The protein mAmetrine-Gp54 was evaluated in terms of binding affinity against P. aeruginosa cells and against other bacterial species to assess its specificity through fluorescence microscopy analysis and spectrofluorimetry assays. Briefly, for microscopy experiments, P. aeruginosa HB10 was used as the target bacteria and E. coli HB106 as the negative control bacteria, and the procedure described previously was conducted [66]. After, bacterial samples were then examined in a confocal microscope LSM780 (Zeiss) using the brightfield 5 mW 488-645 nm light source or under a laser at 405 nm (DPSS 405-10) for mAmetrine excitation and setting a bandpass J o u r n a l P r e -p r o o f filter (500-625 nm) in the Zeiss ZEN 2010 software.…”

“…The target bacteria were then recognized by recombinant fluorescent RBPs. For the detection of P. aeruginosa, a novel RBP (Gp54) was designed and characterized in terms of recognition capabilities and together with the previously described E. coli RBP Gp17 [66] assisted in the development of the novel microfluidic-based method. Overall, our developed assay revealed to be very specific and sensitive, can be completed in 70 minutes, and achieved a detection limit of around 10 3 bacterial cells in blood.…”