Genomic variation in Pseudomonas aeruginosa clinical respiratory isolates with acquired resistance to a bacteriophage cocktail

Whilst virulent bacteriophages have shown promise in treating P. aeruginosa infections, bacteriophage insensitive mutant bacteria (BIMs) have been described. The aim of this study was to examine the genetic changes associated with the bacteriophage insensitive phenotype. Biofilms of 3 genetically distinct P. aeruginosa strains, including ATCC 15692 and 2 clinical respiratory isolates (1 CF and 1 non-CF) were grown for 7 days, and treated with either a cocktail of 4 bacteriophages or a vehicle control for 7 consecutive days. BIMs were detected by streak assays, and resistance to the phage cocktail was confirmed using spot test assays. Comparison of whole genome sequencing between the recovered BIMs and their respective vehicle control-treated phage-sensitive isolates revealed structural variants in 2 strains, and several small variants in all 3 strains. Further analysis of these BIMs showed variants involving TonB-dependent outer membrane receptors in 2 strains, as well as mutations in lipopolysaccharide synthesis genes in 2 strains. We postulate that these outer membrane structures may represent receptor sites for the bacteriophages in the cocktail tested. Prophage deletion and induction were also noted in 2 strains, as well as mutations in several genes associated with virulence factors and antibiotic resistance. These findings may have implications for future applications of lytic phage therapy.