Antistaphylococcal Phage Enhancement by Restriction Bypass and Dynamic Imaging

About This Grant

Project Summary/Abstract Staphylococcus aureus is the most common invasive human pathogen and a major contributor to infection-related morbidity and mortality. Antibiotic resistance is often prevalent at baseline and can develop on therapy. Further compounding the clinical challenge, even susceptible bacteria fail to respond appropriately to first-line therapies in up to 30% of cases. In this context, antibiotic-bacteriophage combination therapies are increasingly considered as potential adjunct or salvage therapies. Unfortunately, the host range of bacteriophage can be limited by DNA restriction-modification systems that efficiently degrade “foreign” DNA, even originating from other S. aureus bacteria. Custom testing of individual isolate-phage combinations is time and labor- intensive, limiting the broad adoption of phage therapy. Additionally, an inability to trace phage particles in situ results in persistent questions about dosing frequency, phage distribution to different body tissues and penetration into infectious foci. Here we address these limitations through optimization of the phage propagation strain. We add methylation specificity proteins to the propagation host to modify the daughter phage DNA. This modified phage DNA is recognized as “self” by a wide variety of S. aureus bacteria, improving the daughter phage host range without any permanent genetic changes to the phage. As an optional enhancement, the propagation strain can make a component of phage capsids fluorescent, enabling tracking of daughter phage for pharmacokinetic and pharmacodynamic studies. This should work for any phage propagated in the enhanced propagation strain; however, subsequent generations infecting other bacteria will lose the fluorescent capsid. To address this challenge, and enable studies including phage amplification in situ and phage penetration into sequestered infection, we will modify the genome of a representative antistaphylococcal bacteriophage, Sb-1, to express the fluorescent capsid regardless of the host strain. With these new tools we will increase the likelihood of phage being able to infect a given isolate and enable visualization of bacteriophage for pharmacokinetic, pharmacodynamic, and pre-clinical analysis.