The growing threat of multi-drug resistant bacteria has meant that the interest in phage therapy as an alternative to antibiotics has likewise been growing. In particular, Pseudomonas aeruginosa is an opportunistic pathogen for which novel treatments are urgently needed, as demonstrated by the World Health Organisation (WHO) priority pathogens list. In this instance, clinical isolates of P. aeruginosa (n = 40) were provided by Mark Reading and Victoria Hargreaves at the Queen’s Veterinary Hospital in Cambridge. The histories of the strains were recorded and confirmed to be P. aeruginosa using both genomic and microbiological approaches. They were then assayed for phenotypic characteristics, including strain competition, motility, growth on minimal media, and antibiotic susceptibility, displaying significant diversity. A subset of strains (n = 15) was then selected based on characteristics that would make them clinically hard to treat, for example: increased biofilm formation or antibiotic resistance, as well as increased virulence. Further interrogation was performed using microbiological and bioinformatic approaches in an effort to document the diversity of the veterinary-derived clinical P. aeruginosa strains.

An important aspect of a phage therapeutic approach is that potential treatment involves a collection of diverse bacteriophages, to reduce the risks of the bacteria developing resistance, as well as bacteriophages with broad host ranges, to ensure that they can clear the target infection. Therefore, bacteriophages (n = 45) were isolated from various natural environments by direct plating and different enrichment strategies involving the lab derived strain, PAO1, and assorted clinical veterinary isolates, with the aim of biasing the selection towards diverse Pseudomonas phages with broad host ranges. The host ranges of these environmental phages were determined amongst the clinical isolates and the phages were categorised by various techniques including transmission electron microscopy (morphology/taxonomy), DNA sequencing and genomics. The analysis revealed a spectrum of phages of varying morphologies and genome sizes, including “jumbo” phages of over 200kb, as well as a definite trend of a greater variety of phages being isolated on clinical, as opposed to the lab, strains.

Representative bacteriophages of the taxonomic families present were selected for more thorough analysis, including genomic and amino acid comparisons of the phages to other known members of the genus. Differences in host ranges were interrogated through comparisons of tail fibre proteins. Bacteriophages were also tested for their ability to clear infection in liquid culture, using the lab strain PA01. Those showing increased ability to clear infection were then selected for experiments looking at their ability to disrupt growth of clinical strains, both in liquid culture and in biofilms. Although phages MSPA46 and MSPA55 showed promise in clearance experiments with PA01, the effect was less pronounced in clinical strains, suggesting further work should look at optimising phage propagation and storage. Previous studies on transducing phages, in Pseudomonas aeruginosa PAO1 and human clinical isolates from cystic fibrosis patients, encouraged investigation into the new environmental phages for any generalised transduction capacity.

Overall, 45 bacteriophages were isolated on 40 clinical strains of P. aeruginosa using a variety of techniques. Both the clinical strains and bacteriophages were interrogated using a combination of microbiological and genomic approaches, which informed a selection process for both to be used in clearance experiments with clinical relevance. Two bacteriophages emerged as promising candidates for future veterinary phage therapy of P. aeruginosa in canine otitis, however further optimisation work is required.