Development of novel bacteriophage-based therapies to combat Pseudomonas aeruginosa and Candida spp. dual-species biofilms. - PROJECT SUMMARY/ABSTRACT
Pseudomonas aeruginosa, Candida albicans, and Candida auris are opportunistic pathogens of great concern
due to their broad arsenal of virulence factors and resistance to antimicrobial drugs. These pathogens are among
the most common agents in hospital-acquired infections and are often co-isolated from lung and wound
infections. Furthermore, these infections are often in the form of mixed-species biofilms, which can provide
additional protection against antimicrobial compounds. The antimicrobial resistance crisis of the last few decades
in combination with the rise of difficulties in discovery of drugs with novel mechanisms of action have resulted in
urgent needs for alternative treatments. Bacteriophage (phage) therapy and antimicrobial peptides (AMPs)
present as encouraging approaches. Phage therapy has been used to combat bacterial infections for over a
century and is an exciting alternative approach to combat drug resistant bacteria. Phage therapy employs
bacterial viruses as antibacterial agents yet provides no remedy for fungal infections. There also are no known
lytic viruses that infect Candida spp. Therefore, the present study aims to develop novel treatments against P.
aeruginosa and Candida spp., using combinations of phages and antimicrobial peptides, with focus on the
treatment of dual-species biofilms. In addition to developing combination treatments utilizing antimicrobial
peptides and phages, we propose to genetically engineer P. aeruginosa phages to produce AMPs to enhance
peptide access to targeted Candida spp. within mixed-species biofilms. This approach alleviates two potential
concerns with the current state of AMP application: low bioavailability due to peptide instability in body fluids and
excessive toxicity if higher systemic doses are required. Dual-species biofilms will be generated in vitro,
subjected to these treatments, and analyzed to determine remaining live biomass using metabolic studies.
Differentially fluorescently labeled strains will also be used for confocal microscopy to determine strain-specific
responses to these treatments. Suitable antimicrobial peptides will be identified against single- and dual-species
populations growing planktonically and in biofilms. Phage engineering will involve using a CRISPR-Cas9 system
to edit phage genomes to encode anti-Candida and anti-biofilm AMPs. The phage engineering approach
provides not only novel alternative treatments, but also a new phage engineering pipeline, expanding the use of
phages as cargo delivery vehicles to infection-specific sites. To our knowledge, this is the first study investigating
engineering phages to produce AMPs to treat mixed-species populations, and thus may also serve as a template
for the treatment of mixed-species infections beyond P. aeruginosa and Candida spp.
