Optimized methods for isolation and characterization of bacteriophage by VT-FACS - Abstract
Antibiotic-resistant (AMR) pathogens are emerging at alarming rates and current treatment options are
becoming increasingly limited, expensive, and in some cases, nonexistent. Two million antibiotic resistant
infections occur annually in the U.S and, although there is an urgent and immediate need for agents with activity
against these emerging multidrug-resistant pathogens, only 2 new antibiotics have been approved since 2009.
Therefore, the development of alternative antibacterial agents is crucial. A promising alternative to antibiotics is
bacteriophage (phage) which have a natural ability to selectively infect and kill target bacteria. Phage is receiving
renewed interest as a safe and effective therapy, but techniques for the isolation, characterization, production
and testing of phages to identify ‘suitable’ phage cocktail candidates are time consuming, expensive and low
throughput. Here we propose innovative high-throughput methodologies based on a proven VT-FACS platform
which mitigate or circumvent these constraints providing time- and cost-savings in development and production
of therapeutic phage cocktails.
The goal of this project is further development of VT-FACS, a high-throughput phage isolation pipeline,
to advance, characterize and support the use and rapid selection of phage as an alternative to classical
antibiotics. Aim 1 will define and validate methods for screening and predicting likelihood of insolating pathogen-
specific phage in prepared and archived lysates for informed on-demand selection. In Aim 2 we will use VT-
FACS and adapt current workflow to identify the optimal bacterial host for production of the most inclusive and
highest titer phage to minimize final cocktail complexity and production cost. Finally, Aim 3 will use VT-FACS to
develop and validate high-throughput methods that isolate phage with antibiofilm activity. Through these 3
independent aims we will develop essential methods which each accelerate development and production of
bacteriophage as an antibacterial therapy. For highest throughput and efficiency in a future working environment,
the proposed methods can be applied in tandem, are inherently scalable and have potential to be automated.
Successful completion of this work will provide expedited and cost-effective means to select and produce
pathogen specific phage with the required host-range and secondary activities necessary for phage therapy. The
ultimate outcome of the research supports improved and personalized phage therapy options to combat today’s
urgent antibiotic resistance threat.
