Developing the predatory bacterium Bdellovibrio bacteriovorus as a novel therapy for treating canine otitis externa

Canine otitis media is a bacterial infection commonly caused by multi-drug resistant Pseudomonas aeruginosa1. Severe cases can result in the only remaining therapeutic option being surgery2,3, which may result in deafness (R. White pers comm). As such, novel approaches to antibacterial therapies are desperately needed. Bacteriophage, viruses that kill bacteria, are relatively well-studied alternatives to antibiotics, however resistance of the bacteria to bacteriophage can arise readily. Other 'living' antibacterials that do not result in such genetically-encoded resistance by the pathogen may represent a better alternative. The predatory bacterium Bdellovibrio bacteriovorus may be one such approach. Bdellovibrio bacteriovorus grows by preying upon susceptible Gram-negative bacteria, including many antibiotic-resistant pathogens, including Pseudomonas spp, E. coli, and Vibrio spp amongst others. We have previously shown that Bdellovibrio can effectively reduce the colonisation levels of Salmonella in chicks4, accompanied by an improvement in clinical symptoms; this has been followed by in vivo infection models involving Shigella5, Klebsiella6, Vibrio7 and Yersinia8 spp. Bdellovibrio are both self-replicating and self-limiting, both advantages over conventional antibiotics. In animal trials, Bdellovibrio does not cause harm8, at worst eliciting a temporary mild inflammation9. Bdellovibrio has been shown to efficiently kill prey growing in biofilms10, and as such it has potential for use against topical infections, such as external ear infections. The aim of this project is to determine the potential efficacy of Bdellovibrio therapy for the treatment of canine otitis externa caused by Pseudomonas aeruginosa, and to study the added potential of combinatorial predator therapy involving both Bdellovibrio and bacteriophage, building upon some of our recently published work11. Main objectives: 1: Quantify the predatory ability of Bdellovibrio to kill in vitro Pseudomonas aeruginosa isolates from cases of canine otitis media, both in pure and mixed cultures, containing other bacterial species co-isolated from the same clinical samples. 2: To build on previous data showing that killing of E. coli by the combination of a Bdellovibrio with a bacteriophage could result in complete eradication of prey in an in vitro assay, to look at the combinatorial effects of Bdellovibrio and either a) a Pseudomonas phage against a pure culture of Pseudomonas, or b) a phage targeting a second bacterial species, against a mixed culture of Pseudomonas and a second species. 3: Assess the efficacy of Bdellovibrio predation on Pseudomonas cells forming biofilm structures in the presence of epithelial cells. Using a published tissue culture-based approach whereby P. aeruginosa forms biofilms on monolayers of epithelial cells12,13, Bdellovibrio predation in the presence of both bacterial prey and epithelial cells will be studied, such as would be encountered during therapy. Together these objectives will reveal the potential efficacy of Bdellovibrio, and combinatorial therapies with bacteriophage in complex, multi-species populations of bacteria. It will inform us as to the best approaches to take in future in vivo therapeutic trials (outside the scope of this project). As Pseudomonas aeruginosa is both an animal and human pathogen, this project may lead to new therapeutic approaches for a variety of infections.