The Application of Bacteriophage and Photoacoustic Flow Cytometry in Bacterial Identification

Infection with resistant bacteria has become an ever-increasing problem in modern medical practice. Bacteremia is a serious and potentially lethal condition that can lead to sepsis without early intervention. Currently, broad-spectrum antibiotics are prescribed until bacteria can be identified through blood cultures, a process that can take 2-3 days and is unable to provide quantitative information. Staphylococcus aureus (S. aureus) is a leading cause of bacteremia, and methicillin-resistant S. aureus (MRSA) accounts for more than a third of the cases. Other bacteria such as Clostridium difficile, Acinetobacter baumannii, and Carbapenem-resistant Enterobacteriaceae are becoming more prevalent and antibiotic-resistant. Rapid diagnostics for each of these superbugs has been a priority for health organizations around the world. Bacteriophages have evolved for millions of years to develop exquisite specificity in target binding using their host attachment proteins. Bacteriophages are viruses that infect bacteria. Bacteriophages use tail spikes, specialized attachment proteins, to bind specifically to their target bacterial cell surface proteins. We use bacteriophages and parts of bacteriophages as specific tags coupled with photoacoustic flow cytometry for the detection and quantification of bacteria. In photoacoustic flow cytometry, laser light is absorbed by particles under flow, and the ultrasound waves generated on the release of the energy are detected. Photoacoustics involves the detection of ultrasound waves resulting from laser irradiation. In photoacoustic flow cytometry, pulsed laser light is delivered to a sample flowing past a focused transducer, and particles that absorb laser light create a photoacoustic response. Bacteria can be tagged with dyed bacteriophage and processed through a photoacoustic flow cytometer where they are detected by the acoustic response. In this chapter, we describe the procedure and methods used to accomplish this. Often the limiting factor for the treatment of patients is the time spent waiting for results. It is our hope that the work presented in this chapter can be a foundation for future work and provide an ability to detect bacterial pathogens in blood cultures. Bacterial plate cultures and Gram staining are nineteenth-century technologies that have been the gold standards for decades, but current trends in resistant bacteria have necessitated a move toward more rapid and quantifiable diagnostic tools.