Potential of bacteriophage proteins as recognition molecules for pathogen detection.

Bacterial pathogens are leading causes of infections with high mortality worldwide having a great impact on healthcare systems and the food industry. Gold standard methods for bacterial detection mainly rely on culture-based technologies and biochemical tests which are laborious and time-consuming. Regardless of several developments in existing methods, the goal of achieving high sensitivity and specificity, as well as a low detection limit, remains unaccomplished. In past years, various biorecognition elements, such as antibodies, enzymes, aptamers, or nucleic acids, have been widely used, being crucial for the pathogens detection in different complex matrices. However, these molecules are usually associated with high detection limits, demand laborious and costly production, and usually present cross-reactivity. (Bacterio)phage-encoded proteins, especially the receptor binding proteins (RBPs) and cell-wall binding domains (CBDs) of endolysins, are responsible for the phage binding to the bacterial surface receptors in different stages of the phage lytic cycle. Due to their remarkable properties, such as high specificity, sensitivity, stability, and ability to be easily engineered, they are appointed as excellent candidates to replace conventional recognition molecules, thereby contributing to the improvement of the detection methods. Moreover, they offer several possibilities of application in a variety of detection systems, such as magnetic, optical, and electrochemical. Herein we provide a review of phage-derived bacterial binding proteins, namely the RBPs and CBDs, with the prospect to be employed as recognition elements for bacteria. Moreover, we summarize and discuss the various existing methods based on these proteins for the detection of nosocomial and foodborne pathogens.

A magnetic nanoparticle-based microfluidic device fabricated using a 3D-printed mould for separation of Escherichia coli from blood.

Herein, A microfluidic device is described, produced with a 3D-printed master mould that rapidly separates and concentrates Escherichia coli directly from whole blood samples, enabling a reduction in the turnaround time of bloodstream infections (BSIs) diagnosis. Moreover, it promotes the cleansing of the blood samples whose complexity frequently hampers bacterial detection. The device comprises a serpentine mixing channel with two inlets, one for blood samples (spiked with bacteria) and the other for magnetic nanoparticles (MNPs) functionalized with a (bacterio)phage receptor-binding protein (RBP) with high specificity for E. coli. After the magnetic labelling of bacteria throughout the serpentine, the microchannel ends with a trapping reservoir where bacteria-MNPs conjugates are concentrated using a permanent magnet. The optimized sample preparation device successfully recovered E. coli (on average, 66%) from tenfold diluted blood spiked within a wide range of bacterial load (102 CFU to 107 CFU mL-1). The non-specific trapping, tested with Staphylococcus aureus, was at a negligible level of 12%. The assay was performed in 30 min directly from diluted blood thus presenting an advantage over the conventional enrichment in blood cultures (BCs). The device is simple and cheap to fabricate and can be tailored for multiple bacterial separation from complex clinical samples by using RBPs targeting different species. Moreover, the possibility to integrate a biosensing element to detect bacteria on-site can provide a reliable, fast, and cost-effective point-of-care device.

Rapid and multiplex detection of nosocomial pathogens on a phage-based magnetoresistive lab-on-chip platform.

Nosocomial or hospital-acquired infections (HAIs) have a major impact on mortality worldwide. Enterococcus and Staphylococcus are among the leading causes of HAIs and thus are important pathogens to control mainly due to their increased antibiotic resistance. The gold-standard diagnostic methods for HAIs are time-consuming, which hinders timely and adequate treatment. Therefore, the development of fast and accurate diagnostic tools is an urgent demand. In this study, we combined the sensitivity of magnetoresistive (MR) sensors, the portability of a lab-on-chip platform, and the specificity of phage receptor binding proteins (RBPs) as probes for the rapid and multiplex detection of Enterococcus and Staphylococcus. For this, bacterial cells were firstly labelled with magnetic nanoparticles (MNPs) functionalized with RBPs and then measured on the MR sensors. The results indicate that the RBP-MNPS provided a specific individual and simultaneous capture of more than 70% of Enterococcus and Staphylococcus cells. Moreover, high signals from the MR sensors were obtained for these samples, providing the detection of both pathogens at low concentrations (10 CFU/ml) in less than 2 h. Overall, the lab-on-chip MR platform herein presented holds great potential to be used as a point-of-care for the rapid, sensitive and specific multiplex diagnosis of bacterial infections.

Bacteriophage-receptor binding proteins for multiplex detection of Staphylococcus and Enterococcus in blood.

Healthcare-associated infections (HCAIs) affect hundreds of millions of patients, representing a significant burden for public health. They are usually associated to multidrug resistant bacteria, which increases their incidence and severity. Bloodstream infections are among the most frequent and life-threatening HCAIs, with Enterococcus and Staphylococcus among the most common isolated pathogens. The correct and fast identification of the etiological agents is crucial for clinical decision-making, allowing to rapidly select the appropriate antimicrobial and to prevent from overuse and misuse of antibiotics and the consequent increase in antimicrobial resistance. Conventional culture methods are still the gold standard to identify these pathogens, however, are time-consuming and may lead to erroneous diagnosis, which compromises an efficient treatment. (Bacterio)phage receptor binding proteins (RBPs) are the structures responsible for the high specificity conferred to phages against bacteria and thus are very attractive biorecognition elements with high potential for specific detection and identification of pathogens. Taking into account all these facts, we have designed and developed a new, fast, accurate, reliable and unskilled diagnostic method based on newly identified phage RBPs and spectrofluorometric techniques that allows the multiplex detection of Enterococcus and Staphylococcus in blood samples in less than 1.5 hr after an enrichment step.

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

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.