Phage-based platforms for the clinical detection of human bacterial pathogens

Abstract

Bacteriophages (phages) have been utilized for decades as a means for uniquely identifying their target bacteria. Due to their inherent natural specificity, ease of use, and straightforward production, phage possess a number of desirable attributes which makes them particularly suited as bacterial detectors. As a result, extensive research has been conducted into the development of phage, or phage-derived products to expedite the detection of human pathogens. However, very few phage-based diagnostics have transitioned from the research lab into a clinical diagnostic tool. Herein we review the phage-based platforms that are currently used for the detection of Mycobacterium tuberculosis, Yersinia pestis, Bacillus anthracis and Staphylococcus aureus in the clinical field. We briefly describe the disease, the current diagnostic options, and the role phage diagnostics play in identifying the cause of infection, and determining antibiotic susceptibility.

Figure 1. The bacterial luxAB reporter genes were integrated into the CDC plague diagnostic phage to create the ϕA1122::luxAB reporter phage. In the presence of Y. pestis, the reporter phage specifically infects the cell and then uses the host transcriptional and translational machinery to express the luciferase enzyme (LuxAB). In the presence of the reduced flavin mononucleotide (FMNH₂), oxygen and exogenously added aldehyde, luciferase catalyzes a complex reaction of which one of the products is light (maximum emission at ~490 nm). The light signal can be detected by a variety of photomultiplier tube and charge-coupled device instruments.

Figure 2. Blood drawn from a patient suspected of MRSA infection/carriage is incubated in BD BACTEC^TM blood culture bottles (Plus Aerobic/F and Plus Anaerobic/F). If cultures are positive for infection, 10 μl of the positive culture is added to each KeyPath^TM test bottle (ID and RS) and incubated at 35°C for 5 h. Both test bottles contain MicroPhage’s proprietary phage cocktail and culture broth; however, RS also contains cefoxitin; a methicillin analog which inhibits bacteriophage amplification in MSSA, but allows amplification to continue if the organism is resistant to methicillin (MRSA). This 5 h incubation period allows phage amplification to proceed only if the target species is present. Six drops of each incubated inoculum is dispensed into the correspondingly labeled lateral flow chamber. Phage amplification is detected via visualization of anti-phage antibodies conjugated to colloidal gold particles present in the lateral flow device. A positive result in ID chamber represents detection of a S. aureus strain, positive result in RS chamber represents MRSA; no visualization in the RS chamber represents MSSA. Adapted from references and .